Immunomodulating tumor necrosis factor receptor 25 (tnfr25) agonists, antagonists, and immunotoxins

ABSTRACT

This document provides novel compositions and methods utilizing immunomodulating agents that can stimulate or indirectly augment the immune system, or can have an immunosuppressive effect. TNFR25 agonists disclosed herein have an anti-inflammatory and healing effect. They can be used, e.g., to treat disease caused by asthma and chronic inflammation, such as inflammatory bowel diseases including ulcerative colitis and Crohn&#39;s Disease. TNFR25 antagonists disclosed herein can inhibit CD8 T cell-mediated cellular immune responses and can, for example, mitigate organ or tissue rejection following a tissue transplantation. TNFR25 agonists disclosed herein represent biological response modifiers that alter the interaction between the body&#39;s cellular immune defenses and cancer cells to boost, direct, or restore the body&#39;s ability to fight the cancer when given with tumor vaccines. TNFR25 specific immunotoxins disclosed herein are also capable of increasing the effectiveness of a chemotherapeutic regimen by depleting a cancer patient of naturally occurring immunosuppressive cells.

PRIORITY

This application is a continuation of U.S. Application Ser. No.16,814,339 filed Mar. 10, 2020 (now U.S. Pat. No. 11,395,846), which isa continuation of U.S. application Ser. No. 15/704,796 filed Sep. 14,2017 (now U.S. Pat. No. 10,624,950), which is a continuation of U.S.application Ser. No. 14/697,007 filed Apr. 27, 2015 (now U.S. Pat. No.9,839,670), which is a continuation of U.S. application Ser. No.13/457,583 filed Apr. 27, 2012 (now U.S. Pat. No. 9,017,679), which is acontinuation of U.S. application Ser. No. 12/534,228 filed on Aug. 3,2009, now abandoned, which is a division of U.S. application Ser. No.11/512,412, filed Aug. 30, 2006, now abandoned, which claims priority toU.S. Application Ser. No. 60/712,084, filed Aug. 30, 2005, all of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to Tumor Necrosis Factor Receptor Super Family 25(TNFR25) agonists, immunotoxins, antagonists and their use in treatingcancer, inflammation and effecting immunosuppression, respectively.

2. Background

Many disorders of the human immune system fall into two broadcategories: those characterized by an attenuated immune response andthose characterized by overzealous immune responses. Immunodeficiency ischaracterized by an attenuated response. There are congenital (inborn)and acquired forms of immune deficiency. Chronic granulomatous disease,in which phagocytes have trouble destroying pathogens, is an example ofthe former. AIDS (“Acquired Immune Deficiency Syndrome”), an infectiousdisease, caused by the HIV virus that destroys CD4+ T cells, is anexample of the latter. An additional disease that may be characterizedby an attenuated immune response is cancer. In contrast to healthyindividuals, cancer patients' immune systems are no longer capable ofeffectively recognizing and/or destroying tumor cells.

Despite high hopes, there are no medications to date that directlyincrease the activity of the immune system. However, biologicaltherapies have recently been used to recruit the immune system, eitherdirectly or indirectly, to fight diseases such as cancer. Monoclonal(MAb) antibodies are now frequently used as a biologic therapy. Forexample, monoclonal antibodies may react with specific types of cancercells, and have direct or indirect antitumor effects.

Tumor vaccines may be employed therapeutically or for prophylaxis afterprimary therapy. Anti-tumor vaccines may need to induce cellularimmunity in the form of tumor-specific cytotoxic T cells of the CD4 orCD8 phenotype. It is thought that effective anti-tumor immunity requiresthe generation and maintenance for long periods of times of suchcytotoxic cells. In addition evidence indicates that the innate arm ofthe immune system must be activated in order to generate effectiveanti-tumor vaccines. Vaccines that enhance or generate humoral responsesproduce antibodies that can be detected over a relatively long period.To be effective, these antibodies need to be capable of targeting cellsurface antigens in live cell assays. Maintaining specific cellularimmune responses to antigen epitopes (adaptive immunity) may requiremore frequent immunizations, although memory cells can sustain theability to respond and rechallenge the immunizing epitope. As such, itwould of substantial benefit to have access to therapies that would becapable of boosting cancer specific cellular immune responses to tumorvaccines.

On the other end of the scale, an overactive immune system figures in anumber of other disorders, particularly autoimmune disorders such aslupus erythematosus, type I diabetes (sometimes called ‘juvenile onsetdiabetes”), multiple sclerosis, psoriasis, rheumatoid arthritis andinflammatory bowel diseases such as Crohn's Disease and ulcerativecolitis (UC). In these, the immune system fails to properly distinguishbetween self and non-self and attacks a part of the patient's own body.Other examples of overzealous immune responses in disease includehypersensitivities such as allergies and asthma.

Suppression of the immune system is often used to control autoimmunedisorders or inflammation when this causes excessive tissue damage.Immunosuppressive medication intentionally induces an immunodeficiencyin order to prevent rejection of transplanted organs. Commonly usedimmunosuppressants include glucocorticoids, azathioprine, methotrexate,cyclosporin, cyclophosphamide and mercaptopurine. In organ transplants,selective T cell inhibition prevents organ rejection, and cyclosporin,tacrolimus, mycophenolate mofetil and various others are used.

T lymphocytes play a central role in regulating immune responses. HelperT cells express the CD4 surface marker and provide help to B cells forantibody production and help CD8 T cells to develop cytotoxic activity.Other CD4 T cells inhibit antibody production and cytotoxicity. T cellsregulate the equilibrium between attack of infected or tumorigenic cellsand tolerance to the body's cells. A disregulated immune attack can leadto autoimmunity, while diminished immune responsiveness results inchronic infection and cancer.

Tumor Necrosis Factor Receptor 25 (TNFR25) also interchangeably referredto herein as Death receptor 3 (DR3), as discussed herein, is a regulatorof T cell function. Death receptor 3 (DR3) (Chinnaiyan et al., Science274:990, 1996) is a member of the TNF-receptor family. It is also knownas TRAMP (Bodmer et al., Immunity 6:79, 1997), wsl-1 (Kitson et al.,Nature 384:372, 1996), Apo-3 (Marsters et al., Curr Biol 6:1669, 1996),and LARD (Screaton et al., Proc Natl Acad Sci USA 94:4615, 1997) andcontains a typical death domain. Transfection of 293 cells with humanDR3 (hDR3) induced apoptosis and activated NF-KB. The cognate ligand forDR3 has recently been identified as TL1A (Migone et al., Immunity16:479, 2002) and has been shown to have costimulatory activity for DR3on T cells through the induction of NF-KB and suppression of apoptosisby expression cIAP2 (Wen et al., J Biol Chem 25:25, 2003). TL1A alsobinds to the decoy receptor 3 (DcR3/TR-6), indicating that fine-tuningof biological TL1A accessibility is of critical importance. Multiplespliced forms of human DR3 mRNA have been observed, indicatingregulation at the post transcriptional level (Screaton et al., Proc NatlAcad Sci USA 94:4615, 1997).

Many TNF-receptor family members have the ability to induce cell deathby apoptosis or induce costimulatory signals for T cell function. Theregulation of these opposing pathways has recently been clarified forTNF-Rl, the prototypic death domain-containing receptor that can causeapoptosis or proliferation of receptor positive T cells (Micheau andTschopp, Cell 114: 181, 2003). NF-KB activation by a signaling complexcomposed of TNF-Rl via TRADD, TRAF2 and RIP induces FLIPL associationwith a second signaling complex composed of TNFRI, TRADD and FADD,preventing caspase 8 activation as long as the NF-KB signaling persists.DR3 has been shown to be able to induce apoptosis in transfected cellsand to induce NF-KB and all three MAP-kinase pathways (Chinnaiyan etal., Science 274:990, 1996; Bodmer et al., Immunity 6:79, 1997; Kitsonet al., Nature 384:372, 1996; Marsters et al., Curr Biol 6:1669, 1996;Screaton et al., Proc Natl Acad Sci USA 94:4615, 1997; Wen et al., JBiol Chem 25:25, 2003). Blocking of NF-KB, but not of MAP-kinase andinhibition of protein synthesis resulted in DR3-mediated cell death,indicating that NF-KB signals mediate anti-apoptotic effects through thesynthesis of anti-apoptotic proteins.

Expression of human DR3 mRNA is pronounced in lymphoid tissues, mainlyin the spleen, lymph nodes, thymus, and small intestine, indicating animportant role for DR3 in lymphocytes. Murine DR3 has been deleted byhomologous recombination in embryonic stem cells (Wang et al., Mol CellBiol 21:3451, 2001). DR3−/− mice show diminished negative selection byanti-CD3 in the thymus but normal negative selection by superantigensand unimpaired positive selection of thymocytes. Mature peripheral Tcells were unaffected by DR3 deficiency. Despite a significant amount ofpreliminary research, the physiological function of DR3 remains poorlycharacterized.

All scientific publications including patent documents cited herein areincorporated by reference in their entirety for all purposes.

SUMMARY OF THE INVENTION

One aspect of the invention relates to an antibody that binds a TumorNecrosis Factor Receptor 25 (TNFR25) antigen and that can act as aTNFR25 agonist. In one embodiment, the antibody is capable of increasingOT-I CD8 cell expansion when cross-primed by gp96-Ig ovalbumin relativeto a control antibody. In a further embodiment, the antibody is purifiedmonoclonal antibody 4C12.

Another aspect of this invention relates to a TNFR25-specific toxincomprising a toxic agent linked to polypeptide that binds the TNRF25receptor. In one embodiment of this aspect, the toxin-comprising portionincludes the monoclonal antibody 4C12 or an immunospecific portion of4C12. In another embodiment, the toxic agent is selected from aradioactive isotope, ricin, abrin, diphtheria toxin, Pseudomonasexotoxin, or metal ion. In a further embodiment, the polypeptide thatbinds the TNRF25 receptor is the TL1A protein or a fragment or variantthereof. In another aspect of the invention, the TNFR25-specific toxinis used in a method of treating cancer in a patient. Specifically, themethod includes depleting a patient of CD4+/CD25+T regulatory cells(Tregs) by providing the patient with the TNFR25-specific toxin and alsoproviding the patient with a chemotherapeutic agent.

Yet another aspect of this invention relates to a method of activatingTNFR25 receptor expressed on a cell comprising contacting the cell witha TNFR25 agonist. The agonist may be selected from a monoclonal antibody4C12; an antibody that binds TNFR25 and that can increase OT-I CD8 cellexpansion when cross-primed by gp96-Ig-ovalbumin relative to a controlantibody; a soluble TL1A protein; an expression vector with anexpression cassette capable of driving the transgenic expression of aTNFR25 agonist antibody; an expression vector with an expressioncassette capable of driving the transgenic expression of a soluble TL1A;or an expression vector with an expression cassette capable of drivingthe transgenic expression of a TNFR25. This method further includesobserving an increase in TNFR25 receptor signaling.

An additional aspect of this invention relates to an antibody that iscapable of acting as a TNFR25 antagonist. In one embodiment, theantibody binds a TL1A and is capable of decreasing OT-I CD8 cellexpansion when cross-primed by gp96-Ig-ovalbumin relative to a controlantibody. In a further embodiment, the antibody is the purifiedmonoclonal antibody L4G6.

A further aspect of the invention relates to a method of inhibitingTNFR25 receptor signaling in a cell. The method includes contacting thecell with a TNFR25 antagonist. The method further includes observing adecrease in TNFR25 receptor signaling.

Another aspect of this invention relates to a tumor vaccine comprising atumor antigen and a TNFR25 agonist as a biological response modifier. Afurther embodiment of this vaccine also includes an adjuvant.

In yet another aspect of this invention, after isolating a tumorspecific antigen, a vaccine comprising a tumor specific antigen and aTNFR25 agonist, is used to immunize a patient against the tumor.

A further aspect of this invention relates to a method of treatingand/or preventing gut inflammation comprising providing a patient inneed thereof a with an effective amount of a therapeutic compositioncomprising a TNFR25 agonist.

A further aspect of this invention relates to a therapeutic compositionfor the facilitation of an organ transplant comprising a TNFR25antagonist and an immunosuppressant. In one embodiment of this aspect,the immunosuppressant is glucocorticoid, azathioprine, methotrexate,cyclosporin, cyclophosphamide, mercaptopurine, tacrolimus ormycophenolate mofetil.

In another aspect of the invention, a TNFR25 antagonist composition isused in a method of transplanting a tissue from a donor into a host.This method includes the steps of obtaining tissue from a donor;providing a host with a TNFR25 antagonist composition; and transplantingthe tissue into the host.

Another aspect of this invention relates to a method of inhibiting theclonal expansion of a population of cognate CD8 T cells. This methodincludes exposing the CD8 T cells to their cognate antigen and exposingthe CD8 T cells to a TNFR25 antagonist. In a further embodiment, thecognate antigen is associated with tissue to be transplanted from adonor into a host.

Another aspect of the invention relates to an isolated TNFR25 antagonistcomprising a polypeptide encoded by a nucleic acid comprising sequencethat hybridizes under stringent conditions to SEQ ID NOs: 4, 5, 6 and/or16, and wherein the sequence encodes an amino acid sequence capable ofbinding a TL1A protein. In one embodiment, the sequence hybridizes understringent conditions to SEQ ID NO: 4. In another embodiment, thesequence hybridizes under stringent conditions to SEQ ID NO: 5. In afurther embodiment, the sequence hybridizes under stringent conditionsto SEQ ID NO: 6. In a further embodiment, the sequence hybridizes understringent conditions to SEQ ID NO: 16. In yet another embodiment, theTL1A is human or mouse TL1A.

Another aspect of the invention relates to a method of treating and/orpreventing lung inflammation comprising providing a patient in needthereof a with an effective amount of a therapeutic compositioncomprising the TNFR25 antagonist comprising a polypeptide encoded by anucleic acid comprising sequence that hybridizes under stringentconditions to SEQ ID NOs: 4, 5, 6 and/or 16, and wherein the sequenceencodes an amino acid sequence capable of binding a TL1A protein.

Another aspect of the invention relates to a method of transplanting atissue from a donor into a host comprising obtaining the tissue from thedonor; providing the host with the TNFR25 antagonist comprising apolypeptide encoded by a nucleic acid comprising sequence thathybridizes under stringent conditions to SEQ ID NOs: 4, 5, 6 and/or 16,and wherein the sequence encodes an amino acid sequence capable ofbinding a TL1A protein; and transplanting the tissue into the host.

Another aspect of the invention relates to a composition comprising apolypeptide encoded by a sequence that hybridizes under stringentconditions to SEQ ID NOs: 3 and/or 7, and wherein the sequence encodesan amino acid sequence capable of binding a TNFR25 receptor protein; anda toxic agent. In one embodiment, the toxic agent is selected from thegroup consisting of a radioactive isotope, ricin, abrin, diphtheriatoxin, Pseudomonas exotoxin, and metal ion.

Another aspect of the invention relates to a method of treating cancer ma patient comprising depleting a patient of CD4+/CD25+T regulatory cells(Tregs) by providing the patient with a composition of the presentdisclosure; and providing a patient with a chemotherapeutic agent.

A further aspect of the invention relates to a method of treating and/orpreventing gut inflammation comprising providing a patient in needthereof a with an effective amount of a composition comprising apolypeptide encoded by a sequence that hybridizes under stringentconditions to SEQ ID NOs: 3 and/or 7, and wherein the sequence encodesan ammo acid sequence capable of binding a TNFR25 receptor protein. Inone embodiment, the inflammation is a result of irritable bowelsyndrome. In another embodiment, the gut inflammation is a result ofCrohn's disease.

A further aspect of the invention relates to a tumor vaccine comprisinga tumor antigen and a polypeptide encoded by a sequence that hybridizesunder stringent conditions to SEQ ID NOs: 3 and/or 7, and wherein thesequence encodes an amino acid sequence capable of binding a TNFR25receptor protein, as a biological response modifier.

Still another aspect of the invention relates to an expression vectorcomprising a nucleic acid sequence that hybridizes under stringentconditions to SEQ ID NOs: 3 and/or 7, and that encodes an amino acidsequence capable of binding a TNFR25 receptor protein.

Yet a further aspect of the invention relates to an expression vectorcomprising a nucleic acid that hybridizes under stringent conditions toSEQ ID NOs: 4, 5, 6 and/or 16, and wherein the sequence encodes an aminoacid sequence capable of binding a TL1A protein.

Additional advantages of the present invention will become readilyapparent to those skilled in this art from the following detaileddescription, wherein only the preferred embodiment of the invention isshown and described, simply by way of illustration of the best modecontemplated of carrying out the invention. As will be realized, theinvention is capable of other and different embodiments, and its severaldetails are capable of modifications in various obvious respects, allwithout departing from the invention. The present invention may bepracticed without some or all of these specific details. In otherinstances, well known process operations have not been described indetail, in order not to unnecessarily obscure the present invention.Accordingly, the drawings and description are to be regarded asillustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the expression of murine TNFR25 in lymph node cells. MFIis indicated by black numbers for isotype control antibody as primaryantibody and in shaded for antiTNFR25. Detection of TNFR25 required atriple sandwich of primary hamster anti-TNFR25 monoclonal antibody,followed by goat anti-hamster biotin and PE-labeled Strept-Avidin. FIG.1B depicts the expression of TNFR25 on activated lymphocytes. Activationof splenocytes was done with immobilized anti-CD3 (5 μg/ml) and antiCD28 (1 μg/ml) or LPS (1 μg/ml) for 24 hours. Cells were gated for CD4or CD8 or B220 positive and 7-AAD negative cells (subscript a stands foractivated CD4, CD8 or B cells). In the histograms, MFI for expression ofTNFR25 on resting splenocytes and on activated splenocytes is shown.FIG. 1C depicts the expression of TNFR25 on thymocytes. Thymocytes weregated on CD4/CD8 double negative, double positive or single positivecells and evaluated for anti-TNFR25 fluorescence.

FIG. 2A depicts splice forms of murine TNFR25. Splice forms wereobtained by RTPCR of mRNA obtained from resting murine splenocytes andmurine cell lines. CRD: cysteine-rich domain; TM: transmembrane domain;DD: death domain. Asterisks: in frame stop codon. In splice forms 1-4the intron between exon 2 and 3 is not spliced out and contains apremature stop codon. Splice forms 1-4 are likely to be nonfunctionalproteins. Murine Δ5 and Δ6 (corresponding to human Δ6 and Δ6,7) lack acomplete transmembrane domain and are predicted to be secreted formsthat could act as soluble decoy receptor for TL1A. Δ5,6 (potentiallycorresponding to human A3) and FL splice forms are studied as transgenesin this report. Δ5,6,8 and Δ5,6,9 (no human homologues) are predicted tobe membrane anchored but lack the death domain and may have alteredsignaling properties or may act as dominant negative splice forms. Thepreferred DN TNFR25 disclosed herein was truncated after the TM domain.FIG. 2B depicts activation-induced alternative splicing of TNFR25. Bothmouse and human TNFR25 are spliced after activation. The human splicingis shown here because splice forms of TNFR25 are separated better insize after gel electrophoresis than murine forms. Splice forms wereconfirmed by sequencing. PBL were isolated by Ficoll-Hypaque gradientcentrifugation. Five million cells were used in each sample and mRNAswere extracted and converted to cDNA using the Invitrogen cDNA synthesiskit. Activation of PBL by PHA (5 μg/ml), immobilized anti-CD3 (5 μg/ml)and soluble anti-CD28 (1 μg/ml), or PMA (10 ng/ml) and ionomycin (400ng/ml) as indicated. The cells were harvested at the indicated timepoints and RT-PCRs were performed; β-actin was used as the internalcontrol. FIG. 2C depicts activation-induced splicing is PKC-dependentand protein synthesis-independent. Freshly isolated PBL cells werestimulated with PMA (10 ng/ml) alone, or ionomycin (400 ng/ml) alone orin combination. PBL were pretreated with H7 (50 μM), or cycloheximide(10 μg/ml) for half an hour, then PMA and ionomycin were added into thecell culture. The cells were harvested after 12 hours for RT-PCRanalysis.

FIGS. 3A-3G illustrate expression and function of TNFR25-transgenesunder the CD2 promoter and enhancer. FIG. 3A depicts expression ofTNFR25 in transgenic mice compared to B6 wild-type mice and isotypecontrol. Inguinal lymph node cells were gated on CD4, CD8, B220, CD11cpositive cells, or NK1.1 positive and CD3 negative cells or NK1.1/CD3double positive cells. The corresponding NK1.1 is indicated. Theexpression profile for FL TNFR25, Δ5,6 TNFR25 and DN TNFR25 wereidentical. FIG. 3B depicts a Western blot of alternatively splicedTNFR25 transfected tumor cells. 50 μg protein from P815 lysatestransfected with three splice forms of TNFR25 were loaded and blottedwith the anti TNFR25 antibody IOD 1. Lane 1: DN TNFR25; lane 2: Δ5,6TNFR25; lane 3: FL TNFR25. The antibody does not detect Δ5,6 TNFR25 inwestern blots. FIG. 3C illustrates reduced cellularity of the CD4 andCD8 positive cells in FL TNFR25-tg lymph node cells and thymocytescompared to w.t. littermates (n=5) *p<0.05; **p<0.01; ***p<0.001. FIG.3D depicts impaired activation-induced proliferation of FL and Δ5,6TNFR25-transgenic cells. Proliferation of purified CD4 or CD8 cells wasmeasured after 3 days of stimulation by thymidine uptake during the last6 hours. Cells were activated in microtiter plates with immobilized antiCD3 (2 μg/ml) with or without soluble anti CD28 (1 μg/ml) or PMA (10ng/ml) and ionomycin (400 ng/ml). Recombinant mouse IL-2 was used at1000 U/ml. FIG. 3E illustrates FL TNFR25 and Δ5,6 TNFR25 activate NF-KB.NF-KB activation was measured in EL4 cells transfected with FL TNFR25(upper panel) or with A5,6 TNFR25 (lower panel) in response to TNFR25triggering. Cells were treated with the agonistic TNFR25 antibody 4C12(5 μg/ml) for 50 min; soluble TL1A was given for 25, 50, or 75 min asindicated in the form of 25% supernatants from TL1A transfected EL4cells; membrane bound TL1A (MTL1A) was given for 50 min by adding TL1Atransfected EL4-cells directly to TNFR25 expressing EL4. Controlsreceived EL4 (untransfected) supernatants for 50 min. Nuclear extractswere prepared and analyzed by EMSA; the arrow indicates activated NF-KB.FIG. 3F depicts primary Th2 biased cytokine production by w.t., FLTNFR25 and Δ5,6 TNFR25-transgenic CD4 T cells. CD4 T cells from spleenswere purified by negative selection and activated with immobilizedanti-CD3 (2 μg/ml) and soluble anti-CD28 (1 μg/ml) for 3 days.Supernatants were collected for cytokine ELISA assays. The figure isrepresentative of three independent experiments. n.s.: not significant;*p<0.05; **p<0.01; ***p<0.001. FIG. 3G illustrates that TNFR25 cancostimulate Th1 or Th2 cytokine production. Cytokine production ofrestimulated FL TNFR25-tg CD4 T cells was determined undernon-polarizing (Th neutral), Th1, or Th2 polarizing conditions. CD4cells were activated with immobilized anti CD3 (2 μg/ml) and solubleanti CD28 (1 μ/ml) alone (Th neutral), or combined with IL-12 (5 ng/ml)and anti-IL-4 (20 μg/ml) for Th1 polarization, or combined with IL-4 (10ng/ml), anti-IFN-γ (10 μg/ml), and anti-IL-12 (10 μg/ml) for Th2polarization for 4 days. The cells were harvested, washed and replatedon anti CD3 for 24 hours and the supernatants collected for cytokineELISA analysis.

FIGS. 4A-4E depict diminished proliferation of TNFR25-transgenic cellsis not due to apoptosis, lack of IL-2 or IL-2 receptor expression. FIG.4A illustrates normal upregulation of IL-2-Ra (CD25) in transgenic CD4and CD8 cells upon activation. After 72-hour activation with immobilizedanti-CD3 and soluble anti-CD28, splenocytes were harvested, washed andstained with anti-CD25-FITC, anti-CDS-PE, and anti-CD4-CY. Upper panelswild type, lower panels Δ5,6 TNFR25-tg cells. FIG. 4B demonstrates thattransgenic cells do not undergo increased apoptosis upon activation. CD4cells were activated with immobilized anti-mouse CD3 (2 μg/ml) withsoluble anti-mCD28 (1 μg/ml) for three days and stained withAnnexin-V-PE and 7-AAD. Annexin-V positive and 7-AAD negative cellsrepresented the apoptotic cells. FIG. 4C illustrates Reduced IL-2production by Δ5,6 TNFR25 transgenic T cells. T cells were purified bynegative selection and activated with immobilized anti-CD3 and solubleanti-CD28 for 3 days. Supernatants were analyzed for IL-2 production byELISA assay; **p=0.0078. FIG. 4D illustrates that dominant negativeTNFR25-transgenic cells are not Th2 polarized in primary response. CD4 Tcells from the spleen were purified by negative selection and activatedwith immobilized antiCD3 (2 μg/ml) and soluble anti-CD28 (1 μg/ml) for 3days. The supernatants were collected for cytokine ELISA assays. Thefigure is representative of three independent experiments. FIG. 4Edepicts the same antibody-isotype response of DN TNFR25-transgenic andw.t. mice. Mice were immunized intraperitoneally with 100 μg DNP-KLH insterile PBS and DNP-specific IgG 1 and IgG2a antibodies in serum wereevaluated by ELISA three weeks after immunization. High-binding 96-wellplates were coated with DNP-BSA at 0.8 μg/ml to detect anti-DNP specificantibodies. Figure represents one of three independent experiments.

FIGS. 5A-5E illustrate that TNFR25 transgenic mice develop a Th2-biasedresponse after in vivo challenge. FIG. 5A demonstrates that anantibody-isotype is Th2 biased in immunized TNFR25-tg mice. Mice wereimmunized intraperitoneally with 100 μg DNP-KLH in sterile PBS withoutadjuvant. DNP-specific IgG 1 and IgG2a antibodies in serum wereevaluated by ELISA one week after immunization. High-binding 96-wellplates were coated with DNP-BSA at 0.8 μg/ml to detect anti-DNP specificantibodies. Data represent one of three independent experiments.**p<0.01; n.s.: not significant. FIG. 5B illustrates TNFR25 signalsregulate eosinophilia in broncho-alveolar fluid (BALF). Immunization andairway challenge of w.t and Δ5,6 TNFR25-tg mice was done as described inExamples 10-15. Airways were lavaged and the differential cell countobtained from Wright-Giemsa stained cytospin preparations. *: p<0.05;n.s.: not significant. FIG. 5C depicts increased lung inflammation inTNFR25-tg mice. Lung histology of mice i.p. sensitized and airwaychallenged with ovalbumin. After bronchial lavage, lungs were removedand fixed in 10% neutral buffered formalin. The lungs were then embeddedin paraffin, cut 5 μm thick, and stained with H&E (left panels) orperiodic acid-Schiff (PAS, right panels) to detect mucus production.Upper two panels wild type B6 mice; lower panels Δ5,6 TNFR25-tg mice.FIG. 5D illustrates that TNFR25 signals control serum IgE levels. Micewere bled on day 0 and three days after the aerosol challenge (day 15).Serum was separated and analyzed by ELISA for ovalbumin specific IgE bysandwich ELISA. Since there is no standard protein available forOVA-specific IgE, results are presented in O.D. units. The figurerepresents one of three independent experiments. **: p<0.01. FIG. 5Edepicts increased Th2 cytokine production by bronchial lymph node cellsin TNFR25-tg mice. Bronchial lymph nodes were harvested one day afteraerosol challenge (day 13) and cells were restimulated in vitro with 100μg/ml ovalbumin for 4 days. Supernatants were then analyzed for cytokineproduction by ELISA. This figure is the representative of twoindependent experiments. *p<0.05; **p<0.01; ***p<0.001

FIGS. 6A-6H illustrate that dominant negative TNFR25 interferes with Th2polarization and lung inflammation. FIG. 6A illustrates that DN TNFR25transgene blocks cytokine costimulation by endogenous TNFR25. W.t. andDN TNFR25-tg CD4 cells were stimulated for three days with anti CD3, orwith the agonistic anti TNFR25 antibody 4C12 and anti CD3 combined andthe supernatants analyzed for cytokines by ELISA. FIG. 6B illustratesthat DN TNFR25 blocks costimulation of proliferation by endogenousTNFR25. In proliferation assays w.t. and DN TNFR25-tg CD4 cells wereactivated with anti CD3, with 4C12, or with the combination of anti CD3and 4C12 for 3 days and thymidine incorporation measured during thefinal 8 hours. FIG. 6C illustrates that DN TNFR25-tg CD4 T cells producediminished Th2 cytokines in secondary activation. W.t. and transgenicCD4 T cells were purified by negative selection and activated withimmobilized anti-CD3 (2 μg/ml) and soluble anti-CD28 (1 μg/ml) for 3days. Cells were then harvested, washed, replated and restimulated withimmobilized anti-CD3 (1 μg/ml) for 2 days. The supernatants werecollected for cytokine ELISA assay. n.s.—not significant; ***p<0.001.FIG. 6D depicts that DN TNFR25-tg CD4 T cells resist Th2 polarization invitro. W.t. and DN TNFR25-tg CD4 cells were purified and activated forfour days with anti CD3 and anti CD28 under neutral (ThN) or Th2polarizing conditions (by adding IL-4 and blocking IFN-γ with antibody).Cells were harvested, washed and replated on anti CD3 for 24 h,supernatants harvested and cytokines analyzed by ELISA. FIG. 6Eillustrates diminished cellular exudation in BALF in DN TNFR25-tg micecompared to w.t. mice. Mice were primed and then aerosol challenged withovalbumin according to the standard protocol; n=5; *: p<0.05. FIG. 6Fdepicts suppression of lung inflammation in DN TNFR25-tg mice afterimmunization and airway challenge. Upper panel: Absence of perivascularinfiltrates in DN TNFR25-tg mice after antigen aerosol exposure. Lowerpanel: Absence of mucus over production and goblet cell hyperplasia inDN TNFR25-tg mice. Lung inflammation was induced as in FIG. 5 byovalbumin immunization and subsequent aerosol exposure. W.t. controlmice had typical inflammation as shown in FIG. 5 . FIG. 6G illustratessuppression of ovalbumin specific IgE production in DN TNFR25-tg mice.Ovalbumin specific IgE in serum was determined by ELISA. **p<0.01. FIG.6H depicts suppression of Th2 but not Th1 cytokine production by DNTNFR25 in bronchial lymph nodes. Bronchial lymph nodes were harvestedone day after aerosol challenge and cells were restimulated with 1001μg/ml ovalbumin for 4 days. Supernatants were then analyzed for cytokineproduction by ELISA. This figure is the representative of twoindependent experiments. n.d.: not detected; n.s.: not significant; *:p<0.05

FIGS. 7A-7J illustrate that TL1A blocking antibodies abrogate lunginflammation and diminish Th2 cytokine production in the lung. FIGS.7A-D depict that anti TL1A antibody L4G6 is a functional antagonist ofTL1A. FIG. 7A, P815 transfectants with FL TNFR25, FIG. 7B, with Δ5,6TNFR25 and FIG. 7C with TL1A stained by flow cytometry with theappropriate antibodies. FIG. 7D, L4G6 blocks TL1A mediated cytotoxicityof TNFR25 transfected cells. Serially diluted soluble TL1A harvestedfrom supernatants of P815-TL1A transfected cells were mixed with51Cr-labeled P815-TNFR25 target cells. Different anti-murine TL1Amonoclonal antibodies were added into the assay and 51 Cr release wasanalyzed five hours later. FIG. 7E illustrates TL IA blocking antibodyL4G6 suppresses mucus production and lung inflammation in vivo in wildtype C57B16 mice. Schema: Schedule of ovalbumin priming, aerosolchallenge and administration of blocking antibody (L4G6) or controlantibody. Lung histology after PAS staining with control IgG (left) andL4G6-IgG (right). Notice the lack of mucus production in L4G6 treatedanimals (arrows point to mucus in mice treated with control IgG). FIG.7F demonstrates diminished cellular exudation in BALF in L4G6 treatedmice compared to control IgG treated animals. *: p<0.05; **: p<0.01;n.s. not significant. FIG. 7G illustrates diminished IL-5 and IL-13production by ovalbumin restimulated bronchial lymph node cells afterTL1A blockade with L4G6. **p<0.01; ***p<0.001. Experimental details asin FIG. 5 . FIG. 7H depicts TL1A expression after aerosol challenge on asubpopulation of CD11c positive cells (arrow) in the lung. Bronchiallymph nodes were harvested before and after ova-aerosol challenge, andCD11c cells analyzed for TL1A expression. All other bronchial lymph nodecells were TL1A negative. FIG. 7I illustrates a lack of TL1A expressionon lymphocytes from bronchial lymph node cells of ovalbumin immunizedmice after airway challenge. Single cell suspensions were stained in atriple sandwich with antiTL1A as primary monoclonal antibody. Cells weregated using the respective labeled antibody as population marker and theTL1A histogram displayed. Anti-TL1A; isotype control. FIG. 7Jillustrates that TL1A is expressed on activated T lymphocytes in vitro.Splenocytes were activated for 24 h with plate bound anti-CD3 or withLPS and then stained with the anti-TL1A triple sandwich as in FIG. 1 andwith the population marker as indicated. B cells are TL1A negative evenafter LPS activation. Gating on the population marker, TL1A expressionon activated cells is shown.

FIG. 8 depicts that TNFR25 signals are required in NKT cells forinduction of lung inflammation. NKT deficient Ja18 knock out mice (Cui,J. et al. Science 278, 1623-6 (1997)) were primed with ovalbumin andalum as in the standard protocol in material and methods. On day 11 themice received by i.v. adoptive transfer 3.1 million partially purifiedw.t. NKT cells or DN TNFR25-tg NKT cells or PBS as indicated. The micewere aerosolized on day 12 with ovalbumin and on day 14 analyzed. W.t.mice served as positive controls for induction of lung inflammation,Jα18 k.o. mice were immunized and ovalbumin aerosolized without adoptivecell transfer as negative controls. The data are for four mice in eachgroup in two independent experiments. Ja are NKT deficient mice (Cui, J.et al. Science 278, 1623-6 (1997).

FIG. 9 illustrates a model for TL1A/TNFR25 mediated triggering of NKTcells and Th2 polarized CD4 cells in the lung. The model depicts thepotential interaction between TL1A and TNFR25 that may contribute toIL-13 production and induction of AHR. Evidence for the need of IL-13production by NKT cells has been provided previously (Akbari, O. et al.Nat Med 9, 582-8 (2003)). The present communication shows the need forTNFR25 signals on NKT cells for lung inflammation, the expression ofTL1A by CD11c+ cells and the costimulation of CD4 Th2 effectors byTNFR25. Since CD4 effectors can express TL1A it is possible thatTL1A/TNFR25 interaction between CD4 and NKT cells provides the molecularlink for their synergy in asthma. In addition other lung associatedcells may express TL1A and help triggering lung inflammation.

FIG. 10 depicts the results of an experiment in which mice received 1million-OT-I-gfp i.v. Two days later (top panel) they were immunizedi.p. with the indicated dose of ovalbumin in PBS; with EG7-gp96-Ig or3T3-ova-gp96-Ig. The amount of gp96-Ig (in ng) secreted within 24 h bythe number of injected cells is indicated on the abscissa (bottompanel); with 3T3-ova—the amount of ovalbumin secreted within 24 h by thenumber of injected cells is indicated on the abscissa. After 5 days OT-Iexpansion was determined by flow cytometry in the peritoneal cavity(PEC) and in the spleen. Frequency of OT-I among CD8 cells was up to 50%in the PEC and up to 8% in the spleen with 3T3-ova-gp96-Ig immunization.

FIG. 11A depicts the results of an experiment in which anti-TNFR25 mAb4C12 acted agonistically and killed TNFR25 transfected EL4 cells, butnot w.t. EL4. In FIG. 11B, 50 μg of agonistic 4C12 or antagonisticanti-TL1A L4G6 or control IgG was given i.p. 24 hand 72 h afterEG7-gp96-Ig immunization. 4C12 caused 8-10 fold increase of OT-Iexpansion and doubling of peritoneal exudate cells (n=4 mice per group).Anti TL1A inhibits CD8 expansion.

FIG. 12 depicts cross priming of CD8 cells by heat shock protein gp96.Vaccine cells (allogeneic or syngeneic) were transfected with gp96-Igand ovalbumin whereupon they secreted gp96-Ig chaperoning ovalbuminpeptides. Gp96 was detected by CD91 and TLR2/4 on DC resulting in theiractivation and engulfment of gp96-Ig with its bound peptides. ActivatedDC up-regulate B7 (independent of CD40-L) and cross-resent gp96-boundpeptides via Kb (in B6 mice). OT-I are gfp expressing, TCR transgenicCD8 cells adoptively transferred to B6 mice specific for Kb-ova. Theirexpansion can be easily measured by green fluorescent protein.

FIG. 13 depicts FoxP3 positive CD4+CD25+ Tregs expressing TNFR25. CD4cells were purified by depleting B cells CD8 cells and monocytes andthen positively selected by magnetic sorting for CD25 and thenactivated.

FIG. 14 depicts a lack of recovery of DN-TNFR25-tg mice from DSS inducedcolitis. 5 mice in each group received 2% DSS in drinking water for 8days and were then restored to normal water.

FIG. 15 shows that TNFR25 signals abolish Treg inhibition.

FIG. 16 shows Gfp-OT-I locating to the mucosa after gp96-Ig immunizationi.p. Mice received 1 million gfp-OT-I i.v. by adoptive transfer. After 2days the mice received 4 million EG7-gp96-Ig (left) or 4 million EG7(right panels) i.p. as stimulus. Four days later the frequency ofgfp-OT-I was analyzed in IEL, Peyer's patches and LPL in addition to theusual analysis of cells in the PEC, in the spleen and in lymph nodes.

FIGS. 17A-17D show SEQ ID NOs: 1-7 and SEQ ID NO: 16 and selected publicdatabase accession numbers.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the invention to provide novel compositions andmethods utilizing immunomodulating agents that can either stimulate orindirectly augment the immune system or in other cases, have animmunosuppressive effect. TNFR25 agonists disclosed herein representbiological response modifiers that alter the interaction between thebody's cellular immune defenses and cancer cells to boost, direct, orrestore the body's ability to fight the cancer when given with tumorvaccines. TNFR25-specific toxic agents disclosed herein are capable ofincreasing the effectiveness of a chemotherapeutic regimen by depletinga cancer patient of naturally occurring immunosuppressive cells. TNFR25agonists disclosed herein can also have a healing effect. They can beused, among other things, to treat disease that caused by chronicinflammation such as inflammatory bowel disease. TNFR25 antagonistsdisclosed herein are capable of inhibiting CD8 T cell-mediated cellularimmune responses and can for example, to treat asthma and mitigate organor tissue rejection following a tissue transplantation.

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

An “antigen” includes any substance that may be specifically bound by anantibody molecule. Thus, the term “antigen” encompasses biologicmolecules including, but not limited to, simple intermediarymetabolites, sugars, lipids, autoacids, and hormones, as well asmacromolecules such as complex carbohydrates, phospholipids, nucleicacids and proteins.

An “antigenic composition” may comprise an antigen (e.g., a peptide orpolypeptide), a nucleic acid encoding an antigen (e.g., an antigenexpression vector), or a cell expressing or presenting an antigen. SeeU.S. Pub. No. 2003/0185840, which is hereby incorporated by reference inits entirety.

An “immunogen” is a macromolecular antigen that is capable of initiatinglymphocyte activation resulting in an antigen-specific immune response.An immunogen therefore includes any molecule which contains one or moreepitopes that will stimulate a host's immune system to initiate asecretory, humoral and/or cellular antigen-specific response.

The term “antibody” encompasses polyclonal and monoclonal antibodypreparations. Antibodies of the invention may be prepared in any mammal,including mice, rats, rabbits, goats and humans. The antibody may be amember of one of the following immunoglobulin classes: IgG, IgM, IgA,IgD, or IgE, and the subclasses thereof, and preferably is an IgG 1antibody.

The term antibody also refers to functional equivalents of theantibodies described in this specification. Functional equivalents havebinding characteristics comparable to those of the antibodies, andinclude, for example, chimerized, hybrid, humanized and single chainantibodies as well as fragments thereof. Methods of producing suchfunctional equivalents are disclosed in PCT Application WO 93/21319,European Patent Application No. 239,400; PCT Application WO 89/09622;European Patent Application 338,745; and European Patent Application EP332,424. Functional equivalents include polypeptides with amino acidsequences substantially the same as the amino acid sequence of thevariable or hypervariable regions of the antibodies of the invention.“Substantially the same” amino acid sequence is defined herein as asequence with at least 70%, preferably at least about 80%, and morepreferably at least about 90% homology to another amino acid sequence,as determined by the PASTA search method in accordance with Pearson andLipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins.

Methods of making polyclonal and monoclonal antibodies are known in theart. Polyclonal antibodies are generated by immunizing a suitableanimal, such as a mouse, rat, rabbit, sheep or goat, with an antigen ofinterest. In order to enhance immunogenicity, the antigen can be linkedto a carrier prior to immunization. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Furthermore, the antigen may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc., in order to enhance the immunogenicity thereof.

Rabbits, sheep and goats are preferred for the preparation of polyclonalsera when large volumes of sera are desired. These animals are gooddesign choices also because of the availability of labeled anti-rabbit,anti-sheep and anti-goat antibodies. Immunization is generally performedby mixing or emulsifying the antigen in saline, preferably in anadjuvant such as Freund's complete adjuvant (“FCA”), and injecting themixture or emulsion parenterally (generally subcutaneously orintramuscularly). The animal is generally boosted 2-6 weeks later withone or more injections of the antigen in saline, preferably usingFreund's incomplete adjuvant (“FIA”). Antibodies may also be generatedby in vitro immunization, using methods known in the art. Polyclonalantisera is then obtained from the immunized animal.

Monoclonal antibodies are generally prepared using the method of Kohlerand Milstein, Nature (1975) 256:495-497, or a modification thereof orCampbell in “Monoclonal Antibody Technology, The Production andCharacterization of Rodent and Human Hybridomas” in Burdon et al., Eds.,Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13,Elsevier Science Publishers, Amsterdam (1985); as well as by therecombinant DNA method described by Huse et al in Science 246, 1275-1281(1989).

Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of non-specifically adherent cells) by applying a cellsuspension to a plate or well coated with the antigen. B-cells,expressing membrane-bound immunoglobulin specific for the antigen, willbind to the plate, and are not rinsed away with the rest of thesuspension. Resulting B-cells, or all dissociated spleen cells, are theninduced to fuse with myeloma cells to form hybridomas, and are culturedin a selective medium (e.g., hypoxanthine, aminopterin, thymidinemedium, “HAT”). The resulting hybridomas are plated by limitingdilution, and are assayed for the production of antibodies which bindspecifically to the immunizing antigen (and which do not bind tounrelated antigens). The selected monoclonal antibody-secretinghybridomas are then cultured either in vitro (e.g., in tissue culturebottles or hollow fiber reactors), or in vivo (e.g., as ascites inmice).

The “antigen-binding site,” or “binding portion” refers to the part ofthe immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions,”or “FRs”. Thus the term “FR” refers to amino acid sequences which arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”

As used herein, the terms “immuno-specific,” “immunological binding” and“immunological binding properties” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and on geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. The ratio of K_(off)/K_(on) enables cancellation ofall parameters not related to affinity, and is thus equal to thedissociation constant K_(d). See, generally, Davies et al. (1990) AnnualRev. Biochem. 59:439-473.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)2” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent VH:: VL heterodimer including an antigen-bindingsite which retains much of the antigen recognition and bindingcapabilities of the native antibody molecule. Inbar et al. (1972) Proc.Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linked VH::VLheterodimer which is expressed from a gene fusion including VH- andVL-encoding genes linked by a peptide-encoding linker. Huston et al.(1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methodshave been described to discern chemical structures for converting thenaturally aggregated-but chemically separated-light and heavypolypeptide chains from an antibody V region into an sFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

“Cognate antigen” as used herein refers to an antigen for which a CD8 Tcell receptor (TCR) is immuno-specific. Such antigen, which aregenerally derived from e.g. pathogen, transplanted tissues (alloantigen)and tumor cells, are recognized by the immune system as non-self.Binding of the cognate antigen to its CD8 T cell receptor results in theclonal expansion of that T cell. A growing population of cognate CD8 Tcells is then in a position to mount a cellular immunological responseagainst the source of the offending cognate antigen.

Antibodies of the invention can also be used to make “immunotoxins.” Thehybrid molecule combines the specificity of an antibody or antigen withthe toxicity of the toxin. As such, immunotoxin molecules have anantigen binding portion and a toxic agent portion. Immunotoxins arepreferably specific for a cell surface molecule, e.g., TNFR25, andfacilitate the delivery of a toxic agent to a cell expressing theaforementioned cell surface molecule.

Preferably, the “toxic agents” have a cytostatic and/or cytotoxic effecton the cell to which it is delivered. Preferred toxic agents are, forexample, radioactive isotopes, fluorescers, chemiluminescers, enzymes,enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, and metalions. Toxic agents include but are not limited to Iodine-131,Indium-111, and Technetium-99m, Technetium-99m, Indium-111, Yttrium-90,doxorubicin (Yang et al. (1988) Proc. Natl. Acad. Sci. USA85:1189-1193), daunorubicin (Diener et al. (1985) Science 231:148-150;Dillman et al. (1988) Cancer Res. 48:6097-6102), methotrexate (Uadia etal. (1985) J Natl Cancer Inst. 74:29-35; Deguchi et al. (1986) CancerRes. 46:3751-3755), and chlorambucil (Symth et al. (1986) J Immunol.137:3361-3366). Other toxic agents include ricin, abrin, diphtheriatoxin and Pseudomonas exotoxin, or an enzymatically active portion (Achains) thereof. See, e.g., U.S. Pat. No. 4,753,894 to Frankel et al.;Nevelle, et al. (1982) Immunol Rev 62:75-91; Ross et al. (1980) EuropeanJ Biochem 104; Vitteta et al. (1982) Immunol Rev 62:158-183; Raso et al.(1982) Cancer Res 42:457-464, and Trowbridge et al. (1981) Nature294:171-173. Also included are enzymatically active toxins and fragmentsthereof such as modeccin, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,and enomycin.

“Toxic agents” can also be chemotherapeutic agents. The term“chemotherapeutic agent” as used herein generally relates to compoundsadministered to stop, slow or reverse unwanted proliferation of cells.Preferably such agents have an anti-proliferative effect.

Chemotherapeutic agents may be anti-metabolites such as 5-FU, forexample. 5-FUbased chemotherapy comprises administration of 5-FU, itsderivatives, alone or with other chemotherapeutics, such as leucovorinor with a DPD inhibitor such as uracil, 5-ethynyluracil,bromovinyluracil, thymine, benzyloxybenzyluracil (BBU) or5-chloro-2,4-dihydroxypyridine. Furthermore, it has been found thatco-administration of a 5′-deoxy-cytidine derivative of the formula (I)with 5-FU or a derivative thereof significantly improves delivery of achemotherapeutic agent selectively to tumor tissues as compared with thecombination of 5-FU or a derivative thereof with a DPD inhibitor5-ethynyluracil.

Alternatively, genotoxic agents are those that form persistent genomiclesions and are preferred for use as chemotherapeutic agents in theclinical management of unwanted cellular proliferation. The rate ofcellular repair of genotoxin-induced DNA damage, as well as the rate ofcell growth via the cell division cycle, affects the outcome ofgenotoxin therapy. A general class of genotoxic compounds that are usedfor treating many cancers are DNA alkylating agents and DNAintercalating agents. Psoralens are genotoxic compounds known to beuseful in the photochemotherapeutic treatment of cutaneous diseases suchas psoriasis, vitiligo, fungal infections and cutaneous T cell lymphoma.Harrison's Principles of Internal Medicine, Part 2 CardinalManifestations of Disease, Ch. 60 (12th ed. 1991). Another general classof genotoxic compounds, members of which can alkylate or intercalateinto DNA, includes synthetically and naturally sourced antibiotics. Ofparticular interest herein are antineoplastic antibiotics, which includebut are not limited to the following classes of compounds representedby: amsacrine; actinomycin A, C, D (alternatively known as dactinomycin)or F (alternatively KS4); azaserine; bleomycin; carminomycin(carubicin), daunomycin (daunorubicin), or 14-hydroxydaunomycin(adriamycin or doxorubicin); mitomycin A, B or C; mitoxantrone;plicamycin (mithramycin); and the like. Still another general class ofgenotoxic agents that are commonly used and that alkylate DNA, are thosethat include the haloethylnitrosoureas, especially thechloroethylnitrosoureas. Representative members of this broad classinclude carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine and streptozotocin. Haloethylnitrosourea first agents can beanalogs or derivatives of any of the foregoing representative compounds.

Tumors currently manageable by platinum coordination compounds such ascisplatin or oxaliplatin include testicular, endometrial, cervical,gastric, squamous cell, adrenocortical and small cell lung carcinomasalong with medulloblastomas and neuroblastomas. Other cytotoxicanti-cancer therapeutic agents include, for example, BEP (bleomycin,etoposide, cisplatin) for testicular cancer, MVAC (methotrexate,vinblastine, doxorubicin, cisplatin) for bladder cancer, MVP (mitomycinC, vinblastine, cisplatin) for non-small cell lung cancer treatment.

Yet another general class of genotoxic agents, members of which alkylateDNA, includes the sulfur and nitrogen mustards. These compounds damageDNA primarily by forming covalent adducts at the N7 atom of guanine.Representative members of this broad class include chlorambucil,cyclophosphamide, ifosfamide, melphalan, mechloroethamine, novembicin,trofosfamide and the like. Oligonucleotides or analogs thereof thatinteract covalently or noncovalently with specific sequences in thegenome of selected cells can also be used as genotoxic agents, if it isdesired to select one or more predefined genomic targets as the locus ofa genomic lesion.

Another class of agents, members of which alkylate DNA, include theethylenimines and methylmelamines. These classes include altretamine(hexamethylmelamine), triethylenephosphoramide (TEPA),triethylenethiophosphoramide (ThioTEP A) and triethylenemelamine, forexample.

Additional classes of DNA alkylating agents include the alkylsulfonates, represented by busulfan; the azinidines, represented bybenzodepa; and others, represented by, e.g., mitoguazone, mitoxantroneand procarbazine. Each of these classes includes analogs and derivativesof the respective representative compounds.

In one embodiment, chemotherapeutic agents are inhibitors of receptortyrosine kinases such as EGFR and HER2-neu and are employed as selectiveinhibitors of the growth of proliferative cells. For example, erbstatin,an EGF receptor tyrosine kinase inhibitor, reduces the growth of EGFRexpressing human carcinoma cells. Various derivatives of styrene arealso stated to possess tyrosine kinase inhibitory properties and to beof use as anti-tumor agents. Two such styrene derivatives are Class IRTK inhibitors whose effectiveness have been demonstrated by attenuatingthe growth of human squamous cell carcinoma injected into nude mice.Certain 4-anilinoquinazoline derivatives are useful as inhibitors ofreceptor tyrosine kinases. The very tight structure-activityrelationships shown by these compounds indicates a clearly-definedbinding mode, where the quinazoline ring binds in the adenine pocket andthe anilino ring binds in an adjacent, unique lipophilic pocket. Three4-anilinoquinazoline analogues (two reversible and one irreversibleinhibitor) have been evaluated clinically as anticancer drugs.Additionally, the monoclonal antibody trastazumab (HERCEPTIN™) for thetreatment of HER2-neu overexpressing metastatic breast cancers.Scheurle, et al., Anticancer Res 20:2091-2096, 2000.

An “expression vector” is any genetic element, e.g., a plasmid,chromosome, virus, behaving either as an autonomous unit ofpolynucleotide replication within a cell. (i.e., capable of replicationunder its own control) or being rendered capable of replication byinsertion into a host cell chromosome, having attached to it anotherpolynucleotide segment, so as to bring about the replication and/orexpression of the attached segment. Suitable vectors include, but arenot limited to viruses, plasmids and cosmids.

Expression vectors contain an “expression cassette” which includes apolynucleotide sequence to be transcribed operably linked topolynucleotide sequences which are necessary to effect ligation orinsertion of the vector into a desired host cell and to effect thetranscription of the polynucleotide to be expressed. Such sequencesinclude promoter sequences to effect transcription, enhancer sequencesto increase transcription, ribosomal binding site sequences andtranscription and translation termination sequences. Alternatively,expression vectors may be capable of directly expressing nucleic acidsequence products encoded therein without ligation or integration of thevector into host cell DNA sequences.

The term “operably linked” refers to the linkage of a DNA segment toanother DNA segment in such a way as to allow the segments to functionin their intended manners. A DNA sequence encoding a gene product isoperably linked to a regulatory sequence when it is ligated to theregulatory sequence, such as, for example, promoters, enhancers and/orsilencers, in a manner which allows modulation of transcription of theDNA sequence, directly or indirectly. For example, a DNA sequence isoperably linked to a promoter when it is ligated to the promoterdownstream with respect to the transcription initiation site of thepromoter, in the correct reading frame with respect to the transcriptioninitiation site and allows transcription elongation to proceed throughthe DNA sequence. An enhancer or silencer is operably linked to a DNAsequence coding for a gene product when it is ligated to the DNAsequence in such a manner as to increase or decrease, respectively, thetranscription of the DNA sequence. Enhancers and silencers may belocated upstream, downstream or embedded within the coding regions ofthe DNA sequence. A DNA for a signal sequence is operably linked to DNAcoding for a polypeptide if the signal sequence is expressed as apreprotein that participates in the secretion of the polypeptide.Linkage of DNA sequences to regulatory sequences is typicallyaccomplished by ligation at suitable restriction sites or via adaptersor linkers inserted in the sequence using restriction endonucleasesknown to one of skill in the art.

“Small molecule” as used herein refers to molecular weight syntheticcompounds designed to interact with a specific protein known to beinvolved in a given disease condition. Libraries of such compounds maybe readily synthesized by combinatorial chemistry for example, andscreened for TNFR25 agonist/antagonist activity using conventionaltechniques.

The terms “TNFR-SF25”, “TNFR25” or “DR3” are all used interchangeablyherein for a member of the TNF receptor family whose complete biologicalfunction was previously not known. See U.S. Pat. No. 6,713,061, andBorysenko, et al., Biochem Biophys Res Commun. 2005 Mar. 18;328(3):794-9, Sheikh, et al., Curr. Cancer Drug Targets. 2004 February;4(1):97-104, which are incorporated by reference in their entirety.However, the inventors, however, have made a number of importantdiscoveries. The cDNA sequence encoding mouse TNFR25 is shown as SEQ IDNO: 1 (FIG. 17A). The cDNA encoding human TNFR25 is shown as SEQ ID NO:2 (FIG. 17B).

Unlike that of any other member of the TNF-R family, DR3 expression wasfound to be controlled by alternative mRNA splicing. Resting T cellsexpress little or no DR3 protein, but contained high levels of randomlyspliced DR3 mRNA. Upon T cell activation via the T cell receptor,protein kinase C (PKC) is activated. PKC activation in turn mediatescorrect splicing of full-length DR3 and surface expression of theprotein. This unique regulation of DR3 expression allows for rapid DR3protein expression on T cells and enables environmental regulation ofDR3 expression via influencing PKC levels responsible for DR3 splicingand expression.

DR3 is also involved in co-stimulating T cell polarization and instimulating the production of IL-13 and IL-10 in Th2 polarized cells.This is an important observation because among other things, IL-10production plays a critical role in suppressing inflammatory boweldisease.

Transgenic expression of TNFR25 in T cells mediates TH2 polarization ofcytokine and antibody production upon T cell activation and antigenexposure. In addition transgenic TNFR25 partially inhibits TCR drivenproliferation of CD4 and CD8 cells and reduced total T cell numbers inlymphoid organs without inducing apoptosis. CD8 cells were more affectedby TNFR25 than CD4 cells. As such, TNFR25 signals are important ineffector responses to pathogens by shaping the ensuing polarizationtowards TH2 or towards a mixed TH1/TH2 response.

TNFR25 transgenic mice are highly susceptible to antigen induced airwayinflammation in an asthma model in mice and produced increasedquantities of IL-13 and eosinophils in the lung upon antigen exposure byinhalation (FIG. 9 ). Transgenic mice expressing a dominant negativeform of TNFR25 showed increased resistance to airway hyperreactivitywhen compared to wild type mice.

“TL1A” is referred to herein as a TNF-like factor that acts as acostimulator of IFN-gamma secretion through binding to the deathdomain-containing receptor, DR3. TL1A, like TNF, is also presumed tocirculate as a homotrimeric soluble form. As such, “soluble TL1A” asused herein, refers to homotrimeric TL1A. The term is not limited to anyspecies specific form. However, the cDNA sequence for the human TL1Amonomer is provided as SEQ ID NO: 7 (FIG. 17D) and that of mouse as SEQID NO: 3 (FIG. 17B).

TL1A has been suggested to play a role in inflammatory bowel disease(IBD) by functioning as a Th1-polarizing cytokine. It has been shownthat the amount of TL1A protein and the number of TL1A-positive cellscorrelate with the severity of inflammation, most significantly inCrohn's Disease (CD). It has also been shown that the addition ofrecombinant human TL1A to cultures of PHA-stimulated lamina propriamononuclear from CD patients significantly augmented IFN-gammaproduction by 4-fold, whereas a minimal effect was observed in controlpatients. Additionally, a blocking anti TL1A antibody was able toameliorate asthma in wild type mice indicating that TNFR25 and TL1A areinvolved in the pathogenesis of asthma.

The receptors of the human body work by being stimulated or inhibited bynatural (such as hormones, cytokines and neurotransmitters) or synthetic(such as drugs, e.g., antibodies or small molecules) agonists.

“TNFR25 agonist” is referred to herein as a substance that binds to theTNFR25 receptor and triggers a response in the cell on which the TNFR25receptor is expressed similar to a response that would be observed byexposing the cell to a natural TNFR25 ligand, e.g., TL1A. An agonist isthe opposite of an antagonist in the sense that while an antagonist mayalso bind to the receptor, it fails to activate the receptor andactually completely or partially blocks it from activation by endogenousor exogenous agonists. A partial agonist activates a receptor but doesnot cause as much of a physiological change as does a full agonist.

Soluble TL1A might be given in therapeutic form to a patient to increasethe activation of the TNFR25 receptor in a given cell population as aTNFR25 agonist.

Alternatively, another example of a TNFR25 agonist is an antibody thatis capable of binding and activating TNFR25. For example, the monoclonalantibody 4C12 binds and activates TNFR25 signaling. In anotherembodiment and in the context of this invention, a TNFR25 agonist may bederived from an expression vector with an expression cassette capable ofectopically driving the transgenic expression of a TNFR25 agonistantibody and/or TL1A protein at a chosen location or at a chosen time.In yet another embodiment, a TNFR25 agonist leading to an increase inTNFR25 signaling in a tissue is provided by an expression vector with anexpression cassette capable of driving the transgenic expression ofTNFR25 itself. This would be useful in a situation where increasedTNFR25 signaling is desired in a tissue in which there is an excess ofexogenous or endogenous TNFR25 agonist(s) relative to receptor.

“TNFR25 antagonist” is referred to herein as a substance that inhibitsthe normal physiological function of a TNFR25 receptor. Such agents workby interfering in the binding of endogenous receptor agonists/ligandssuch as TL1A, with TNFR25 receptor. An example of a TNFR25 antagonist isa dominant negative TNFR25 receptor. Preferably, the TNFR25 antagonistused herein is an antibody specific to TL1A which interferes with TL1A'sability to activate the TNRF25 receptor. Most preferably, that antibodyis the monoclonal antibody L4G6. In another embodiment, the TNFR25antagonist is a fusion protein of the extracellular portion of TNFR25 oran alternative splice form of TNFR25 with The Fe portion ofimmunoglobulin or any other suitable fusion partner. In anotherembodiment, the TNFR25 antagonist is a soluble form of TNFR25 made bytruncation above the transmembrane binding domain, either as alternativesplice form or as an artificial construct. In another embodiment, theTNFR25 antagonist is an antibody that specifically binds TNFR25 andinterferes with its binding to its natural ligand(s). In anotherembodiment, TNFR25 antagonist may be an expression vector with anexpression cassette capable of driving the transgenic expression of anantisense mRNA, RNAi or ribozyme that is capable of knocking downendogenous TNFR25 and/or TL1A mRNA transcription and/or translation at achosen location or at a chosen time. In yet another embodiment, one maydecrease TNFR25 signaling in a tissue by providing an expression vectorwith an expression cassette capable of driving the transgenic expressionof a dominant negative TNFR25.

TNFR25 antagonists or agonists may be in the form of aptamers.“Aptamers” are DNA or RNA molecules that have been selected from randompools based on their ability to bind other molecules. In one embodiment,aptamers specifically bind TNFR25 to block binding of its naturalligand, e.g., TL1A, or which bind TL1A itself, and prevent it frombinding TNFR25. In another embodiment, aptamers specifically bind theTNFR25 receptor and activate it.

“Dominant negative” or “DN” as used herein refers to an exogenouslyprovided structural variant of TNFR25 that acts to block endogenousTNFR25. For example, a molar excess of a DN will out compete endogenousTNFR25 for binding of the TNFR25 ligand, e.g., TL1A. Preferably the DNis the same as the wild type TNFR25 except that it is missing theintracellular domain. Alternatively, the DN is the same as the wild typeTNFR25 except that it is missing the transmembrane and the intracellulardomain. The coding sequence for the mouse DN TNFR25 is provided in SEQID NO: 4 (FIG. 17C). The coding sequence for the human DN TNFR25 isprovided in SEQ ID NO: 5 (FIG. 17C). The coding sequence for the humanDN TNFR25 containing only the extracellular domain is provided in SEQ IDNO: 6 (FIG. 17C).

The invention also involves the coding sequences that are substantiallyidentical to SEQ ID NOs: 3-7 (FIGS. 17B-D). The skilled artisan willalso appreciate that oligonucleotide sequences substantially identicalto SEQ ID NOs: 3-7 may differ from SEQ ID NOs: 3-7, respectively, withrespect to the identity of at least one nucleotide base. However, alloligonucleotides sequences substantially identical to SEQ ID NOs: 3-7will hybridize under stringent conditions (as defined herein) to all ora portion of the complements of SEQ ID NOs: 3-6 (i.e., targetsequences), respectively. The terms “hybridize(s) specifically” or“specifically hybridize(s)” refer to complementary hybridization betweenan oligonucleotide (e.g., a primer or labeled probe) and a targetsequence. The term specifically embraces minor mismatches that can beaccommodated by reducing the stringency of the hybridization media toachieve the desired priming for the PCR polymerases or detection ofhybridization signal.

Under stringent hybridization conditions, only highly complementary,i.e., substantially identical nucleic acid sequences, hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving 3 or more mismatches out of 20 contiguous nucleotides, morepreferably 2 or more mismatches out of 20 contiguous nucleotides, mostpreferably one or more mismatch out of 20 contiguous nucleotides. Thehybridizing portion of the hybridizing nucleic acid is at least about90%, preferably at least about 95%, or most preferably about at leastabout 98%, identical to the sequence of a target sequence, or itscomplement.

Hybridization of a nucleic acid to a nucleic acid sample under stringentconditions is defined below. Nucleic acid duplex or hybrid stability isexpressed as a melting temperature (Tm), which is the temperature atwhich the probe dissociates from the target DNA. This meltingtemperature is used to define the required stringency conditions. Ifsequences are to be identified that are substantially identical to theprobe, rather than identical, then it is useful to first establish thelowest temperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g. SSC or SSPE). Then assuming that1% mismatching results in a 1° C. decrease in Tm, the temperature of thefinal wash in the hybridization reaction is reduced accordingly (forexample, if sequences having >95% identity with the probe are sought,the final wash temperature is decrease by 5° C.). In practice, thechange in Tm can be between 0.5° C. and 1.5° C. per 1% mismatch.

Stringent conditions involve hybridizing at 68° C. in 5×SSC/5×Denhart'ssolution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.Moderately stringent conditions include washing in 3×SSC at 42° C. Theparameters of salt concentration and temperature may be varied toachieve optimal level of identity between the primer and the targetnucleic acid. Additional guidance regarding such conditions is readilyavailable in the art, for example, Sambrook, Fischer and Maniatis,Molecular Cloning, a laboratory manual, (2nd ed.), Cold Spring HarborLaboratory Press, New York, (1989) and F. M. Ausubel et al eds., CurrentProtocols in Molecular Biology, John Wiley and Sons (1994).

“Immunosuppressants” as the term is used herein are important drugsnecessary for the treatment of inflammatory diseases and facilitation oforgan transplants. For example, Cyclosporin A (CsA) has considerableimmunosuppressive activity. It has revolutionized organ transplantationand is commonly used in the treatment of autoimmune diseases. For arecent review of the use of CsA and its mechanisms of action, see Wengeret al; Cyclosporine Chemistry, Structure-activity relationships and Modeof Action, Progress in Clinical Biochemistry and Medicine, Vol. 2, 176(1986). However, CsA is a powerful medication that can have severe sideeffects such as renal failure, bone marrow suppression and infertility.

Corticosteroids are also used in inflammatory conditions for theiranti-inflammatory effects. They have a rapid onset of action, andprofoundly affect many parts of the immune system as well as most otherbody systems. Corticosteroids are a cornerstone of treating most typesof vasculitis, and are often used in combination with otherimmunosuppressive medications. However, long term use of corticosteroidcan also have severe side effects including hypertension, weight gain,acne, and a swollen face. Other immunosuppressives include azathioprine,methotrexate, cyclophosphamide, mercaptopurine, tacrolimus andmycophenolate mofetil.

As a general matter, the agonists and antagonists may be provided to apatient in either compositions that are to be swallowed, injected,inhaled or provide by way of suppositories. Alternatively, thecompositions may be formulating into ear or eye drops.

The term “asthma” as used herein includes any asthmatic condition markedby recurrent attacks of paroxysmal dyspnea (i.e., “reversibleobstructive airway passage disease”) with wheezing due to spasmodiccontraction of the bronchi (so called “bronchospasm”). Asthmaticconditions which may be treated or even prevented in accordance withthis invention include allergic asthma and bronchial allergycharacterized by manifestations in sensitized persons provoked by avariety of factors including exercise, especially vigorous exercise(“exercise-induced bronchospasm”), irritant particles (pollen, dust,cotton, cat dander) as well as mild to moderate asthma, chronic asthma,severe chronic asthma, severe and unstable asthma, nocturnal asthma, andpsychologic stresses. The methods of this invention may be particularlyuseful in preventing the onset of asthma in mammals e.g., humansafflicted with reversible obstructive disease of the lower airwaypassages and lungs as well as exercise-induced bronchospasm. In methodsfor treating asthma disclosed herein, the preferred method of deliveringthe inventive antagonists is through inhalation

There are several different types of devices which use generallydifferent mechanisms and methodologies to produce aerosols forinhalation. The most commonly used device is a metered dose inhaler(MDI) which comprises a drug formulation container with the formulationincluding a low boiling point propellant. The formulation is held in thecontainer under pressure and a metered dose of formulation is releasedas an aerosol when the valve on the container is opened. The low boilingpoint propellant quickly evaporates or “flashes” when the formulation isexposed to atmospheric pressure outside the container. The particles offormulation containing the drug without the propellant are inhaled intothe patient's lungs and thereafter migrate into the patient'scirculatory system. There are a number of different types of MDIdevices. Devices of this type are disclosed in U.S. Pat. No. 5,404,871issued Apr. 11, 1995 and U.S. Pat. No. 5,364,838 issued Nov. 15, 1994.

Another type of device is the dry powder inhaler (DPI) device. Asindicated by the name such devices use formulations of dry powder whichpowder is blown into an aerosolized cloud via a burst of gas. TypicalDPI devices are shown in U.S. Pat. No. 5,775,320 issued Jul. 7, 1998 andU.S. Pat. No. 5,740,794 issued Apr. 21, 1998.

Yet another type of aerosol delivery device forces a formulation througha porous membrane. Formulation moving through the pores breaks up toform small particles which are inhaled by the patient. Devices of thistype are shown in U.S. Pat. No. 5,554,646 issued Aug. 13, 1996 and U.S.Pat. No. 5,522,385 issued Jun. 4, 1996.

With respect to inhalable compositions, suitable carrier materials maybe in the form of an amorphous powder, a crystalline powder, or acombination of amorphous and crystalline powders. Suitable materialsinclude carbohydrates, e.g., monosaccharides such as fructose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, trehalose, cellobiose, and the like; cyclodextrins,such as 2-hydroxypropyl-β-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; (b) amino acids, suchas glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine,and the like; (c) organic salts prepared from organic acids and bases,such as sodium citrate, sodium ascorbate, magnesium gluconate, sodiumgluconate, tromethamine hydrochloride, and the like; (d) peptides andproteins, such as aspartame, human serum albumin, gelatin, and the like;(e) alditols, such as mannitol, xylitol, and the like. A preferred groupof carriers includes lactose, trehalose, raffinose, maltodextrins,glycine, sodium citrate, tromethamine hydrochloride, human serumalbumin, and mannitol.

Such carrier materials may be combined with the inventive agonists orantagonists prior to spray drying, i.e., by adding the carrier materialto the buffer solution which is prepared for spray drying. In that way,the carrier material will be formed simultaneously with and as part ofthe inventive agonist or antagonist particles. Typically, when thecarrier is formed by spray drying together with the inventive agonistsor antagonists, the inventive agonists or antagonists will be present ineach individual particle at a weight percent in the range from 5% to95%, preferably from 20% to 80%. The remainder of the particle willprimarily be carrier material (typically being from 5% to 95%, usuallybeing from 20% to 80% by weight), but will also include buffer(s) andmay include other components as described above. The presence of carriermaterial in the particles which are delivered to the alveolar region ofthe lung (i.e., those in the requisite size range below 10 μm) has beenfound not to significantly interfere with systemic absorption ofinventive agonists or antagonists.

Alternatively, the carriers may be separately prepared in a dry powderform and combined with the dry powder inventive agonists or antagonistsby blending. The separately prepared powder carriers will usually becrystalline (to avoid water absorption), but might in some cases beamorphous or mixtures of crystalline and amorphous. The size of thecarrier particles may be selected to improve the flowability of theinventive agonists or antagonists powder, typically being in the rangefrom 25 μm to 100 μm. Carrier particles in this size range willgenerally not penetrate into the alveolar region of the lung and willoften separate from the inventive agonists or antagonists in thedelivery device prior to inhalation. Thus, the particles which penetrateinto the alveolar region of the lung will consist essentially ofinventive agonists or antagonists and buffer. A preferred carriermaterial is crystalline mannitol having a size in the above-statedrange.

The dry powder compositions of the present invention are preferablyaerosolized by dispersion in a flowing air or other physiologicallyacceptable gas stream in a conventional manner. One system suitable forsuch dispersion is described in copending application Ser. No.07/910,048, which has been published as WO 93/00951, the fulldisclosures of which are incorporated herein by reference.

Sterile injectable solutions are prepared by incorporating thecompositions and formulations of the invention in the required amount inthe appropriate solvent, such as sodium phosphate-buffered saline,followed by filter sterilization. As used herein, “a physiologicallyacceptable carrier” includes any and all solvents, dispersion media,antibacterial and antifungal agents that are non-toxic to humans, andthe like. The use of such media and agents for pharmaceutically activesubstances is well known in the art. The media or agent must becompatible with maintenance of proper conformation of the inventiveprotein chains, and its use in the therapeutic compositions.Supplementary active ingredients can also be incorporated into thecompositions. A description of exemplary pharmaceutically acceptablecarriers and diluents, as well as pharmaceutical formulations, can befound in Remington's Pharmaceutical Sciences, a standard text in thisfield, and in USP/NF.

The dosage and mode of administration of claimed compositions should beadjusted according to the identity, formulation, route of administrationand other relevant characteristics pertaining, as is known, or readilydiscernable in the art.

2. TNFR25 Agonists as Direct Potentiators of T Cell Polarization andLung Inflammation

TNFR25 and its ligand TL1A were discovered to be direct potentiators ofT cell polarization and lung inflammation. The important role of TL1Aand TNFR25 in lung disease is supported by the exaggerated lunginflammation observed in agonistic TNFR25-transgenic mice.Over-expression of TNFR25 results in a strong Th2 bias of CD4 cells uponactivation. This bias in turn is likely to be responsible for increasedlung inflammation in the ovalbumin model.

The inventors observed that in a murine model of asthma, blockade ofTNFR25 signaling results in reduced Th2 cytokine production whichinhibits lung inflammation. TNFR25 on CD4 cells triggers IL-13 secretionon both Th1 and Th2 polarized cells and TNFR25 signals are needed inNKT-cells that initiate and promote lung inflammation. Chronic IL-13production, triggered by TNFR25, also is responsible for the sequaelaeof chronic lung inflammation including but not restricted to chronicasthma, for airway remodeling and fibrosis. Blockade of TNFR25 signalingon NKT cells by a dominant negative TNFR25 mutant in adoptive transferexperiments abrogates lung inflammation. Blockade of TNFR25 signals suchas this would be useful in the treatment of acute and chronic asthma andother lung disorders.

In the chain of events leading to lung inflammation, IL-13 production byNKT cells is an early step (Akbari, O. et al. Nat Med 9, 582-8 (2003)).The critical role for NKT cells in asthma is supported by this studysince the adoptive transfer experiments indicated that one of theprincipal molecular switches used by NKT cells to induce lunginflammation is TNFR25. TNFR25 has the additional ability to modulateincipient effector responses by antigen specific CD4 memory cellsthrough PKC mediated TNFR25 splicing in TCR activated cells. TNFR25 thusacts in the very early phase of the initiation of the memory response inthe lung. Therefore, blockade of TNFR25 signaling by anti-TL1A or byother procedures interrupts the cascade of events leading to acute lunginflammation which is thought to be responsible for asthma attacks.

Blockade of TNFR25 was achieved in wild type mice genetically as well asby antibody blockade with the two independent methods giving similarresults. Interfering with TNFR25 signals in vivo resulted in diminishedIL-13, IL-5 and IL-4 production by antigen restimulated drainingbronchial lymph nodes and in suppression of lung inflammation.Importantly, anti TL1A antibody blockade during the phase of airwayantigen challenge of primed and Th2 polarized mice was effective ininhibiting lung inflammation indicating a direct role of TNFR25 in theeffector phase in the lung.

3. Constitutive TNFR25 Expression on NKT Cells and Inducible Expressionon Activated T Cells

To study the biological functions of TNFR25 and its cognate ligand TL1A,hamster anti-mouse monoclonal antibodies were generated by standardprotocols. To reliably detect the low level of TNFR25 expression by flowcytometry on primary cells it was necessary to develop a triple layersandwich assay. Without activation TNFR25, was detected at low levels onnaive CD4 T cells and at even lower levels also on CD8 T cells, but noton B cells (FIG. 1A). In addition a subpopulation of CD 11c+ cellsexpressed TNFR25. NKT cells constitutively expressed relatively highlevels of TNFR25, while only a small fraction of CD3 negative NK11+cells showed TNFR25 expression (FIG. 1A). In the thymus single positiveCD4 and CD8 cells expressed TNFR25 similar to peripheral T cells. CD4,CD8 double positive and double negative thymocytes did not expressTNFR25 (FIG. 1C).

Upon activation of peripheral T cells with anti-CD3 and anti-CD28,TNFR25 expression was upregulated on both CD4+ and CD8+ cells (FIG. 1B).LPS activation of B cells on the other hand did not result in TNFR25expression. Murine TNFR25 mRNA is constitutively expressed in T cellsbut randomly spliced similar to human TNFR25 (Screaton, G. R. et al.Proc Natl Acad Sci USA 94, 4615-9. (1997)) (FIG. 2A). Increased TNFR25protein expression on activated T cells was associated with activationinduced splicing of full length TNFR25 from randomly spliced TNFR25mRNA. Activation induced splicing of TNFR25 in T cells was blocked bythe chemical inhibitor H7 indicating a role for PKC in splicing (FIG.2C).

The preferential expression of TNFR25 on NKT and activated T cells andits activation induced splicing to full length TNFR25 and rapid increasein surface expression raised the question of the biological function ofTNFR25 in the immune system. The ablation of the TNFR25 gene in mice didnot reveal a definitive phenotype for TNFR25 deficiency, except for amild defect in negative selection in the thymus. Hence, the biologicaleffects of T cell-expressed TNFR25-transgenes driven by the CD2 promoterand enhancer were analyzed.

Full length TNFR25 (FL TNFR25, in FIG. 2A) and an alternative spliceproduct of TNFR25 lacking exon 5 and 6 (Δ5,6 TNFR25, FIG. 2A) were usedfor transgenic expression. Δ5,6 TNFR25 lacks exon 5 and 6 encoding partof the fourth cysteine rich, extracellular domain. It is anchored in themembrane, however, and has a complete intracellular signaling domainlike FL TNFR25, and binds TL1A and an agonistic anti TNFR25 antibody(4C12). In addition a dominant negative mutant, DN TNFR25, truncatedimmediately after the transmembrane domain was expressed as transgeneand will be described below.

4. Transgenic Over-Expression of TNFR25 Promotes Th2 Polarization of CD4Cells and Mediates Increased Lung Inflammation in the Ovalbumin Model ofAHR

Four independent founders for each TNFR25 transgene were obtained andanalyzed. The CD2 promoter and enhancer supported position independenttransgene expression in all founders Δ5,6 TNFR25, FL TNFR25 and DNTNFR25 revealed high level expression in resting T cells, NKT cells, NKcells and in a subpopulation of CD11c+ cells. B cells did not expressthe transgene (FIG. 3A). The authenticity of antibody detection by flowcytometry was verified by Western blots of transfected tumor cells (FIG.3B) and identical Western blot bands were detected on transgenicsplenocytes while the endogenous molecule was below the level ofdetection by Western blot. The Δ5,6 splice form was not detected by theantibody 10D1 in Western blots (FIG. 3B) indicating that it binds toexon 5 or 6. Transgenic over-expression of FL TNFR25 was associated withdiminished numbers of T cells in primary and secondary lymphoid organscompared to non-transgenic litter mates (FIG. 3C); the effect of theΔ5,6 transgene on cellularity was modest in the thymus and notsignificant secondary lymphoid organs. The reduced number of T cells intransgenic mice was accompanied by diminished proliferation in responseto anti CD3 and CD28 stimulation when comparing equal numbers ofpurified transgenic with non-transgenic littermate CD4 and CD8 cells(FIG. 3D). Diminished proliferation was seen at all time points from 24to 72 hours. However, stimulation of TNFR25-tg CD4 or CD8 cells with thephorbolester PMA and the Ca-ionophore ionomycin restored normalproliferation indicating that transgenic cells did not have an intrinsicdefect in their ability to proliferate. CD3/CD28 activated TNFR25-tg Tcells upregulated CD25 normally but produced only about one half theamount of IL-2 (FIG. 4C) compared to littermate controls. Exogenousaddition of excess IL-2 did not restore proliferation (FIG. 3D).TNFR25-tg T cells did not undergo increased apoptosis as measured byannexin V staining (FIG. 4B) indicating that the proliferative deficitis not due to TNFR25 signals for cell death.

FL and Δ5,6 TNFR25 transfected EL4 cells were used to compare thesignaling properties of the splice variants triggered with soluble TL1A,membrane bound TL (EL4-TL1A) or an agonistic anti TNFR25 antibody(4C12). All three ligands rapidly induced NF-KB activation within 25 minas detected by EMSA (FIG. 3E).

After primary activation with plate bound anti-CD3 and soluble anti-CD28FL and Δ5,6 TNFR25 transgenic cells produced significantly increasedquantities of Th2 cytokines including IL-4, IL-5, IL-13 and IL-10 whencompared to non-transgenic littermates (FIG. 3F). IFN-γ was diminishedwhen FL TNFR25-tg CD4 cells were activated but not with Δ5,6 TNFR25transgenes, indicating a subtle difference in the function of the splicevariants. Although proliferation of TNFR25-transgenic CD4 cells wasdiminished, increased Th2 cytokine production was detectable alreadywithin 24 hours of activation and continued to increase in the followingdays, indicating Th2 bias existed prior to activation.

Next it was determined whether the Th2 bias of FL TNFR25-transgenic CD4cells could be overruled under Th1 polarizing conditions. Under Thneutral conditions TNFR25-tg CD4 cells produced as much IL-4 as w.t. CD4cells under Th2 polarizing conditions (FIG. 3G). Incubation of FLTNFR25-tg cells under Th2-polarizing conditions had no additional effecton IL-4 or IL-13 production indicating that the cells were alreadymaximally Th2 polarized during primary activation under Th-neutralconditions. However, FL TNFR25-tg CD4 cells could be polarized to Th1 byincluding in the culture antibodies to IL-4 and adding exogenous IL-12.Under these conditions FL TNFR25-tg cells produced IFN-γ at higherlevels than Th1 1-polarized wild type cells (FIG. 3G) indicating thatthe TNFR25 transgene can also costimulate Th1 cytokines. Th1 polarizedFL TNFR25-tg CD4 cells, unlike w.t. Th1 cells, also produced IL-13 butonly minimal amounts of IL-4. Transgenic over expression of TNFR25 whilespontaneously biased towards Th2, nonetheless can costimulate either Th1or Th2 type cytokine production under appropriate polarizing conditions.In addition TNFR25 signals costimulate IL-13 production under either Th1or Th2 polarizing conditions.

The spontaneous Th2 bias of TNFR25-tg mice in vivo by immunization andanalysis of antibody isotype production was evaluated. In vivo studieswere carried out with Δ5,6 TNFR25-transgenic mice. Δ5,6-TNFR25 shows thesame signaling properties as FL TNFR25 with regard to NF-KB induction(FIG. 3E) and induction of apoptosis and generates a similar Th2 biasedcytokine profile. However, unlike the FL-transgenic mice, Δ5,6transgenic mice have normal CD4 T cell cellularity in lymph nodes (FIG.3C) and spleens and therefore may more accurately represent TNFR25function for in vivo experiments.

DNP-KLH immunized TNFR25-tg mice generated increased ratios of antigenspecific IgG 1/IgG2a antibody compared to non-transgenic litter mates,indicative of an increased Th2 type antibody ratio in vivo (FIG. 5A).Without immunization, IgG1 and IgG2a levels of w.t. and transgenic micewere identical, evidencing that activation is necessary to reveal theTh2 bias.

The Th2 bias of TNFR25-tg CD4 cells and their increased IL-13 productionsuggested that TNFR25 overexpression may predispose to increasedallergic lung inflammation characteristic for asthma, as IL-13 is thesignature cytokine of that condition (Elias, J. A. et al. Am J RespirCell Mol Biol 28, 401-4 (2003). This hypothesis was tested using theclassical ovalbumin model for experimental lung inflammation in mice.TNFR25-tg B6 mice and w.t. controls were primed i.p. with ovalbumin andalum on day 0 and S. On day 12 they were airway challenged withaerosolized ovalbumin and analyzed one to three days later. TNFR25-tgmice contained dramatically increased numbers of eosinophils in thebroncho-alveolar fluid (BALF, FIG. 5B), associated with increasedovalbumin specific IgE levels in serum (FIG. 5D) and elevated Th2cytokine production by ovalbumin restimulation of bronchial lymph nodecells (FIG. 5E). IFN-γ production was diminished in transgenic bronchiallymph node cells compared to w.t. cells. Histopathological analysis ofthe lungs of TNFR25-tg mice showed massively increased perivascular lunginfiltration by eosinophils, increased bronchial mucus production andgoblet cell hyperplasia stained with PAS (FIG. 5C) consistent withexacerbation of lung inflammation by TNFR25 over-expression on T cells.

5. Genetic or Antibody Blockade of TNFR25 During Airway Challenge ofPrimed Mice Blocks Lung Inflammation

A dominant negative mutant of TNFR25, DN TNFR25 was made to block TNFR25signaling during airway challenge, and expressed the construct astransgene under the CD2 promoter and enhancer. The DN TNFR25-transgenelacks the entire intracellular signaling domain but is identical to fulllength TNFR25 in its transmembrane and extracellular domain.

Transgenic DN TNFR25 was expressed at identical levels as the agonisticTNFR25 transgenes (FIG. 3A) as determined by flow cytometry. Usingsurface fluorescence intensity as measure for the number of expressedmolecules, a three to four fold molar excess of transgenic TNFR25expression over endogenous TNFR25 was determined. This level ofoverexpression of DN TNFR25 silenced the activity of endogenous TNFR25.Primary anti CD3 activation of both w.t. and DN-transgenic CD4 cellsstimulates both Th1 and Th2 cytokine secretion (FIG. 4D) Triggering ofTNFR25 with an agonistic antibody (4C12) during primary anti CD3activation of w.t cells costimulates both Th1 and Th2 cytokineproduction but this costimulatory effect of TNFR25 is blocked by the DNTNFR25 transgene (FIG. 6A), indicating the transgene blocks the functionof the endogenous gene. Similarly, the agonistic effect of 4C12costimulates proliferation of w.t. CD4 cells which was blocked in DNTNFR25-tg CD4 cells (FIG. 6B) indicating that endogenous TNFR25 issilenced. Importantly, the expression of DN TNFR25 on CD4 cells greatlydiminished the normally observed, upregulated Th2 cytokine productionupon secondary activation of cells that had been primed under Th-neutralconditions (FIG. 6C), indicating that TNFR25 signals strongly promote orare required for Th2 polarization and that these signals are blocked bythe DN-transgene. More importantly, DN TNFR25-tg cells could not be Th2polarized even under Th2 polarizing conditions which were provided bythe addition of IL-4 and blocking antibodies to IFN-γ and IL-12 (FIG.6D). Th1 polarization of DNTNFR25-tg CD4 cells on the other hand was notaffected even under nonpolarizing (Th neutral, ThN) conditions. Thusalthough TNFR25 costimulates the production of both Th1 and Th2cytokines in primary activation of CD4 cells, TNFR25 signals appear tobe necessary to promote Th2 polarization in vitro. Th1 polarization isindependent of TNFR25, but TNFR25 signals in Th1 cells promote IL-13production (FIG. 3G). The absence of TNFR25 signals in DN TNFR25-tg micein vivo does not affect the normal IgG 1 to IgG2a antibody ratio foundin w.t. mice following immunization of DN TNFR25-tg mice with DNP-KLH(FIG. 4E).

Next, it was determined whether DN TNFR25-tg mice had an alteredresponse in the lung inflammation model. Compared to w.t. mice, DNTNFR25-tg mice upon airway challenge exhibited significantly diminishedeosinophilic infiltration in the BALF, absent lung inflammation anddiminished IgE production in serum when examined by histopathology andPAS staining (FIGS. 6E-6G). Restimulating bronchial draining lymph nodesfrom airway challenged DN TNFR25 tg mice with ovalbumin showeddiminished Th2 cytokine production but normal IFN-γ production (FIG.6H). The data suggest that TNFR25 plays a critical role in pulmonaryimmune responses.

To validate the genetic data of TNFR25 blockade in w.t. mice, monoclonalantibodies to murine TL1A were developed. Using TNFR25 and TL1Atransfected cells (FIGS. 7A and 7B), TL1A blocking antibodies thatabrogated TNFR25 signaling were identified. TL1A transfected cellsexpress TL1A on their surface (FIG. 7C) and secrete TL1A into thesupernatant similar to other TNF superfamily members. TL1A containingsupernatants caused rapid 51Cr release from FL TNFR25 or Δ5,6 TNFR25transfected tumor cells through apoptosis as reported previously(Chinnaiyan, A. M. et al. Science 274, 990-2. (1996); Kitson, J. et al.Nature 384, 372-5. (1996); Screaton, G. R. et al. Proc Natl Acad Sci USA94, 4615-9. (1997); Bodmer, J. L. et al. Immunity 6, 79-88. (1997);Marsters, S. A. et al. Curr Biol 6, 1669-76. (1996); Tan, K. B. et al.Gene 204, 35-46 (1997)). (FIG. 7D). One of the anti-TL1A antibodies,L4G6, completely blocked TL1A mediated lysis of TNFR25-transfectedcells, while several other anti-TL1A antibodies either had no effect ormediated only incomplete inhibition of lysis (FIG. 7D). L4G6 antibodytherefore was selected for use of in vivo blockade of TL1A to blockTNFR25 signaling in genetically unmodified wild type mice. Mice wereimmunized twice with ovalbumin in alum as usual. One day prior to airwaychallenge and for the next three days thereafter 50 μg L4G6 was injectedi.p. and then the mice were analyzed (FIG. 7E). Controls received thesame amount and schedule of hamster IgG. L4G6 administered in this wayduring and after the period of aerosol challenge phase inhibitedeosinophil exudation into BALF, blocked excessive mucus production anddiminished the Th2 cytokine production of bronchial lymph node cellsupon rechallenge with ovalbumin in vitro (FIGS. 7E-7G).

Blockade of lung inflammation by anti-TL1A during aerosol challenge isevidence of TL1A expression in the airways. It was found that modestlevels of TL1A are expressed on a subpopulation of CD 11c+ cells inbronchial draining lymph nodes after airway challenge but not prior toairway challenge (FIG. 7H). All other cell populations in bronchiallymph nodes were TL1A negative before and after aerosol challenge (FIG.7I). Inguinal lymph nodes did not express TL1A on CD11c+ cells or anyother cell type at any time before or after ovalbumin priming or airwaychallenge. TL1A expression, however, can be induced in vitro within 24 hon purified CD4+ and CD8+ spleen or lymph node cells by activation withanti-CD3 and anti-CD28 (FIG. 7J). LPS activated, proliferating B cellsdo not express TL1A (FIG. 7J).

6. DN TNFR25-Transgenic NKT Cells Fail to Support Lung Inflammation inAntigen Primed and Aerosol Challenged NKT Deficient Mice

It has been shown that adoptive transfer of w.t. NKT cells to NKTdeficient mice restores lung inflammation and airway hyper reactivity inthe ovalbumin model and that IL-13 production by NKT cells is required(Akbari, O. et al. Nat Med 9, 582-8 (2003); Lisbonne, M. et al. JImmunol 171, 1637-41 (2003); Meyer, E. H. et al. Proc Natl Acad Sci USA103, 2782-7 (2006)). NKT cells have also been implicated in thepathophysiology of asthma patients (Sen, Y. et al. J Immunol 175,4914-26 (2005). Akbari, O. et al. N Engl J Med 354, 1117-29 (2006)). Todetermine whether TNFR25, which is constitutively expressed on NKT cells(FIG. 1A), is involved in triggering lung inflammation, transferred wildtype and DN TNFR25-tg NKT cells were adoptively transferred intoovalbumin primed NKT-deficient mice (Cui, J. et al. Science 278, 1623-6(1997)) (Ja18 k.o.) (FIG. 8 ). While adoptively transferred w.t. NKTcells restored lung inflammation upon airway antigen challenge, the samenumber of DN TNFR25-tg NKT cells was unable to do so. The datademonstrate that TNFR25 signals in NKT cells are critical for triggeringlung inflammation during airway antigen exposure of sensitized mice.

7. TNFR25 Agonists as Direct Potentiators of Anti-Tumor Immune Responses

Mature dendritic cells carry out an important process referred to as“crosspresentation” that enables them to effectively prime T-cytotoxiccells that are specific for tumor-specific peptides. Tumor antigens aredegraded and are presented on MHC Class I proteins to circulating CD8 Tcells.

To demonstrate that TNFR25 agonists are effective tumor vaccine BRMs,mice were injected with EG7 tumor cells and OT-I cells. OT-I cells werethen observed for clonal expansion. EG7 are EL4 mouse ascites lymphomalymphoblast cells that have been genetically altered to expressovalbumin, the major protein constituent of chick egg white. Mice wereinoculated with EG7 cells and received an adoptive transfer ofovalbumin-specific T-cell receptor transgenic cells (OT-I). OT-I cellsact as indicator cells in vivo by responding to the tumor specificovalbumin antigen. Theoretically, the mouse's dendritic cells presentthe tumor-specific ovalbumin to and activate the ovalbumin-specific(OT-I) T cells. However, under these circumstances and as is often seenin human tumor vaccine trials, OT-I cells react with anergy to the EG7tumor cells.

In contrast, co-transfecting EG7 cells with a construct that encodes asecreted version of the heat shock protein gp96 (gp-96-Ig) provided fora tumor secreting gp961g and containing ovalbumin as surrogate antigen.Exposure of the CD8 OT-I cells to the secreted chaperone heat shockprotein in combination with the tumor specific ovalbumin resulted in anexpansion of OT-I from an initial frequency of 0.5% to over 50% of allCD8 cells following primary injection and one boost with gp961gsecreting. Therefore, cross priming of CD8 cells by gp96-Ig-ovalbumincompared to cross-priming by intact ovalbumin protein is enhanced 10,000to 1,000,000 fold. See Example 19 and FIGS. 10 and 12 . See also Am JReprod Immunol. 2002 October; 48(4):220-5.

When OT-I CD8 cells were cross-primed by gp96-Ig-ovalbumin, in thepresence of the TNRF25 agonist antibody 4C12, OT-I CD8 expansionincreased by an additional IO-fold over a control antibody. However,when OT-I CD8 cells were cross-primed by gp96-Igovalbumin, in thepresence of the TL1A blocking antibody L4G6, OT-I CD8 expansion wasdecreased by an 10-fold over a control antibody. See FIG. 11B.

As such, TNFR25 agonists are effective biological response modifiers fortumor vaccines because they boost T cell activation and the cellularimmune response to a tumor specific antigen, whereas TNFR25 antagonistsblocked or inhibited T cell activation. Therefore, another aspect of theinvention relates to methods and therapeutic agents that increase theeffectiveness of a tumor vaccine.

Tumor vaccines attempt to the use of elements of the body's naturalimmune system to fight cancer. Tumor vaccines contain one or more tumorspecific antigens and may contain an adjuvant and biological responsemodifiers. A tumor specific antigen is a polypeptide that issubstantially limited to expression in or on tumor cells and which canbe used to stimulate an immune response intended to target those tumorcells. Different types of vaccines are used to treat different types ofcancer. For an antigenic composition to be useful as a vaccine, anantigenic composition must induce an immune response to the antigen in acell or tissue. As used herein, an “antigenic composition” may comprisean antigen (e.g., a peptide or polypeptide), a nucleic acid encoding anantigen (e.g., an antigen expression vector), or a cell expressing orpresenting an antigen. See U.S. Pub. No. 2003/0185840, which is herebyincorporated by reference in its entirety.

Biologic response modifiers (BRM), which have been shown to upregulate Tcell immunity or downregulate suppressor cell activity. Such BRMsinclude, but are not limited to, Cimetidine (CIM; 1200 mg/d)(Smith/Kline, Pa.); low-dose Cyclophosphamide (CYP; 300 mg/m2)(Johnson/Mead, N.J.), cytokines such as g-interferon, IL-2, or IL-12 orgenes encoding proteins involved in immune helper functions, such asB-7.

In one embodiment of this aspect of the invention, the tumor vaccinecomposition includes a tumor antigenic composition and a TNFR25 agonist.In another embodiment, the TNFR25 agonist is the antibody 4C12. In thepreferred embodiment, a TNFR25 agonist is added to a tumor vaccine as abiological response modifier. Even more preferably, the TNFR25 agonistis the antibody 4C12. In another embodiment the tumor vaccine includesan adjuvant.

Tumor vaccine adjuvants may include IL-1, IL-2, IL-4, IL-7, IL-12,gamma-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and norMDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables. Either as liquid solutions orsuspensions: solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

8. TNFR25 Immunotoxins as Indirect Potentiators of Innate Anti-TumorImmune Responses

Many scientists and corporations work on CD4+/CD25+T regulatory cells(Tregs) because of their immense potential impact on the treatment ofmany diseases. Although many people are exposed to the sameenvironmental allergens and sensitization to allergens is common, only afraction of people develop allergic diseases such as asthma. The reasonfor this is unclear at present but could be related to the presence ofefficient regulatory Tregs that suppress airway inflammation in healthyallergen exposed individuals. As such, Tregs are known contribute to themaintenance of peripheral tolerance against self and non-self. However,Tregs have also been documented to impede the body's ability to fightcancer. In those cases, Tregs interfere with the body's tumor-killingimmune cells. As such, Tregs function as dedicated suppressor cells andmay play a role in preventing tumors, e.g., in squamous cell carcinomaof the head and neck, from being recognized by the immune system. See BrJ Cancer, 2005 Mar. 14; 92(5):913-20.

The Inventors have observed that Tregs have properties that suggest thatthey are activated. Other TNF receptors have been reported to beexpressed on Tregs. GITR is expressed by activated Tregs. Its ligationhas been found to abolish the inhibitory activity of Tregs (Nocentini etal., Eur J Immunol. 2005 April; 35(4):1016-22). TNFR25 has manyproperties that could make it a versatile regulator of Tregs cells. a)TNFR25 protein expression is rapidly upregulated by PKC induced mRNAsplicing; b) Several functional splice versions including a decoyreceptor and a dominant negative form allow fine tuning of regulation;c) TL1A expression appears to be highly regulated on Crohn's Disease; inaddition TL1A expression on activated lymphocytes allows sensing oflymphocyte density. Finally, they exert their regulatory function atleast in part by secreting IL-10 and IL-13, a cytokine triggered byTNFR25 signals. The inventors are the first to note that FoxP3expressing, CD4+/CD25+ cultured Tregs express inordinately high TNFR25levels. As such, the Inventors conclude that TNFR25 modulates Tregfunction and that TNFR25 can be used as a molecular tag to deplete Tregsin vivo. See Example 20. Moreover, TNFR25 agonists abolish Treginhibition. See FIG. 15 .

Therefore, another aspect of the invention relates to methods andtherapeutic agents that are useful in increasing the potency ofanti-cancer therapies by depleting a patient of CD4+/CD25+T regulatorycells (Tregs). In one embodiment of this aspect of the invention, animmunotoxin used to deplete Tregs. In this embodiment, the immunotoxinhas an antigen binding portion that is specific for TNFR25; and isconjugated to a toxic agent. In an alternate embodiment, the patient isprovided with soluble TL1A conjugated to a toxic agent. Yet anotherembodiment relates to a chemotherapeutic composition having achemotherapeutic agent and a TNFR25-specific immunotoxin. Still anotherembodiment relates to a chemotherapeutic composition having achemotherapeutic agent and a TL1A conjugated to a toxic agent.

Another aspect of the invention relates to methods and therapeuticagents that are useful in increasing the potency of anti-cancertherapies by providing a patient with TNFR25 agonists reduce inhibitionmediated by CD4+/CD25+T regulatory cells (Tregs).

9. TNFR25 Agonists as Anti-Inflammatory Agents

Using a dominant negative form of TNFR25 (DN-TNFR25) lacking theintracellular death domain and an alternatively spliced form of TNFR25(delta 5,6-TNFR25) lacking exon 5 and 6 encoding the fourthextra-cellular cysteine rich domain, the Inventors found that TNFR25function is required to restore homeostatic balance after a mucosalinsult. Specifically, the inventors transgenically expressed a dominantnegative form of TNFR25 (DNTNFR25) under the CD2 promoter in mice. Themice were given dextran sodium sulfate (DSS) to induce colitis as amodel for human Crohn's disease. Wild type C57B 1/6 mice developedcolitis and diarrhea and lost weight after 5 days of drinking water with2% DSS. However, if restored to normal water, wild type mice recoveredwithin a week and regained weight, whereas DN-TNFR25-tg expressing miceacquired disease in a similar manner to wild type mice but diarrheaappeared more severe. Additionally, restoration to normal water did notresult in their recovery. Instead all DN-TNFR-tg expressing micecontinued to lose weight and died within the second week. Miceexpressing functional transgenes of TNFR25 (full length TNFR25 or asplice version Δ5,6-TNFR25) recovered in a manner similar to wild typemice. See Examples 21 and 22 and FIG. 14 .

Current treatment of Crohn's disease uses anti-inflammatory agents,immunosuppressives, and TNF inhibitors. All these are symptomatic.Stimulating TNFR25 signaling moves one step up in the pathogenetic chainof events. Activated TNFR25 stimulates IL-10 and IL-13 production,which, in turn, stimulates TGF-beta production resulting in restorationof homeostasis. Such a treatment is curative or approaching cure.

Therefore, another aspect of the invention relates to a method oftreating inflammatory bowel disease in a patient by administering to apatient a therapeutic amount of a TNFR25 agonist. Another embodimentrelates to a method of treating inflammatory bowel disease in a patientby raising IL-10 levels in the mucosal areas in the intestine. Inanother embodiment, the TNFR25 agonist is the antibody 4C12. In afurther embodiment, the TNFR25 agonist is a soluble form of TL1A. In thepreferred embodiment, the inflammatory bowel disease is Crohn's disease.

Given this anti-inflammatory activity of TNFR25 agonists, it is anotheraspect of the invention to provide methods and therapeutic agents to apatient requiring reduction of inflammation. In one embodiment, apatient is provided a composition containing a TNFR25 agonist todecrease inflammation and promote healing. In one embodiment, a patientis provided with a composition containing the TNFR25 agonist 4C12. Suchembodiments are useful for alleviating the symptoms of disordersmediated by chronic inflammatory responses at the cellular level,including cardiovascular diseases (e.g., atherosclerosis), autoimmunediseases including systemic lupus erythematosis (SLE), multiplesclerosis (MS), diabetes (especially type I diabetes), ankylosingspondulitis, arthritis (particularly rheumatoid arthritis), asthma andallergy, bone resorptive disorders, opthalmological disorders includingretinopathies, and fibrotic diseases.

10. TNFR25 Antagonists as Immunosuppressives

Many organs and tissues are now routinely transplanted from one human toanother. Except for the rare cases where the donor and recipient aremonozygotic “identical” twins, such grafts are called allografts. Tissuematching for the transplantation of tissues from one individual toanother is critical because a tissue recipient will mount a stronghumoral and cellular immune reaction against all non-self proteins.Tissue typing involves identifying MEW antigens on both donor andrecipient cells and using donor cells with as many MHC alleles identicalto those of the recipient as possible. Matching MEW Class I (especiallyHLA-B) and Class II HLA-DR alleles is more important for successfultransplantation than matching other MHC antigens; and matching MEW ismore important than matching minor histocompatibility antigens.

HLA matching improves graft survival but does not prevent rejection,even in MHC-identical siblings (except for identical twins). AllogeneicMHC is recognized by either CD8 T cells (Class I) or CD4 T cells (ClassII); up to 10% of T cells can recognize a given allogeneic MEW becauseit resembles self MHC+foreign peptide.

Improved success in transplantation is due to growing technicalexpertise, increasing availability of transplant centers to do HLAmatching and minimize organ delivery time, and the increasedavailability of immunosuppressive drugs (cyclosporin and tacrolimus)that block T cell activation to alloantigens. Still problematic areshortages of organs, the ability of existing disease to destroy thetransplanted organ (diabetes and HBV infection are two examples), sideeffects of immunosuppressive drugs and high cost.

Given the major side effects associated with current immunosuppressivesthat block T cell activation and the observation that TNFR25 antagonistssuch as TL1A blocking antibody L4G6, make effective inhibitors ofcognate CD8 T cell clonal expansion (see FIG. 11B, Example 19), anotheraspect of the invention involves the use of TNFR25 antagonists for thefacilitation of tissue transplantation to prevent tissue rejection. Inone embodiment, TNFR25 antagonists are provided to a transplantrecipient to suppress the clonal expansion of CD8 T cells that carryalloantigen-specific T cell receptors (TCRs) and to relieve suppressionof Tregs by TNFR25. In another embodiment, TNFR25 antagonists areprovided to a transplant recipient in combination with animmunosuppressive agent.

EXAMPLES Example 1 Media and Reagents

Cells were cultured in Iscove's Modified Dulbecco's Minimal EssentialMedium (Invitrogen) supplemented with 10% heat-inactivated FBS(Invitrogen), 10 μg/ml gentamycin (Invitrogen), and 50 μMβ-mercapto-ethanol (Bio-Rad). Monoclonal anti-mouse CD3 and antihumanCD3 were purified from culture supernatants of the 2C 11 and the OKT3cell lines, respectively (ATCC, Manassas, Va.). Monoclonal anti-mouseCD28 and anti-human CD28 were purchased from eBioscience (San Diego,Calif.). ConA, PHA, and LPS were purchased from Sigma (St. Louis, Mo.).Recombinant murine IL-2 was from BioSource International (Camarillo,Calif.). PMA, ionomycin, H7, and cycloheximide were purchased fromCalbiochem (San Diego, Calif.).

Directly conjugated monoclonal antibodies, including FITC-CD4,CychromeCD4, PE-CD8a, Cychrome-CD8a, FITC-B220, PE-B220, FITC-CD25,PE-CD11c, PEDX5, FITC-CD3, PE-NK1.1, PE-Annexin V and 7-AAD werepurchased from BD/PharMingen (San Diego, Calif.). Hamster IgG controlwas purchased from eBioscience.

Example 2 Generation of Monoclonal Antibodies Against mTNFR25 and mTL1A

Armenian hamsters were immunized intraperitoneally three times biweeklywith 50 μg of mTNFR25-Ig or mTL1A-MBP (maltose binding protein) inFreund's adjuvant. Three days prior to the fusion, hamsters were boostedwith 50 μg of the respective proteins intravenously. Hamster splenocyteswere fused with the murine myeloma SP20 with PEG and then plated inmethylcellulose-based medium for two weeks using ClonaCell-HY kit(StemCell Technologies Inc., BC, Canada). One thousand colonies werepicked and analyzed by ELISA in plates coated with the immunizing fusionprotein or control protein-Ig fusion protein. Supernatants from positiveclones were tested for the ability to detect mTNFR25 isoforms intransfected cells by flow cytometry and western blotting. Antibodieswere purified from a Nutridoma-SP (Roche, Indianapolis, Ind.)supernatant on a protein G column, dialyzed into PBS and filtersterilized.

Example 3 Flow Cytometric Analysis for the Expression of mTNFR25 andmTL1A

Single cell suspensions were prepared from lymphoid organs indicated inthe individual experiment. Prior to staining, cells were treated withpurified anti-mouse CD16/CD32 (Fc-γIII/II receptor; BD) and purifiedhuman IgG (Jackson ImmunoResearch, West Grove, Pa.) to blocknon-specific binding to FcRs. Cells were stained with Armenian hamsteranti-mouse TNFR25 or anti-mouse TL1A for 30 minutes at 4° C. Cells werewashed in FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) and thenstained with biotin-labeled goat anti-Armenian hamster IgG (JacksonImmunoResearch) for 30 minutes at 4° C. Cells were washed and thenstained with Streptavidin-PE or Streptavidin-Cychrome (BD) for 30minutes at 4° C. Cells were washed and then stained with directlyconjugated cell surface markers for distinct cell populations. Sampleswere analyzed using a Becton Dickinson F ACS LSR instrument andCELLQUEST™ software.

Example 4 RT-PCR

Identification of splice forms of mTNFR25: Messenger RNA was extractedfrom murine cell lines or tissues with the Micro Fast-Track kit(Invitrogen, Carlsbad, Calif.) and cDNA was reverse transcribed usingthe Superscript II kit (Invitrogen). RT-PCR products were subcloned intothe PCR II vector using the TOPO cloning kit (Invitrogen) and wereconfirmed as splice forms of mTNFR25 by DNA sequencing. For splicinganalysis of murine TNFR25 the following primers were used. Upstreamprimer, exon 2: CAG TGA GTC CCA GAA GAG GT (SEQ ID NO: 8); downstreamprimer, exon 7: GGA TAG CCC CAA AAA GGA AC (SEQ ID NO: 9); upstreamprimer, exon 7: TCC TTT TTG GGG CTA TCC TG (SEQ ID NO: 10); downstreamprimer, exon 10: GGT ATT TCT CCA TGA CGC TT (SEQ ID NO: 11).

Activation-induced alternative splicing of human TNFR25 was analyzedbecause in the mouse the PCR products of different splice forms weremore difficult to distinguish on agarose gels but appeared similar tohuman splice forms. The following PCR primers were used. Upstream exon4: TTC ACC CTT CTA CTG CCA AC (SEQ ID NO: 12); downstream, exon 7: TAACCA GGG GCT TGT GAG GC (SEQ ID NO: 13). Human peripheral bloodmononuclear cells were isolated from healthy donors by Ficoll Hypaquedensity gradient centrifugation. Five million cells per sample wereactivated with PHA (5 μg/ml), or immobilized anti-CD3 (OKT3, 5 μg/ml)and anti-CD28 (1 μg/ml), or PMA (10 ng/ml) and ionomycin (400 ng/ml).The cells were harvested at the indicated time points and mRNA wasextracted and converted to cDNA using the Invitrogen kit. Human β-actinwas used as internal control. Quantitation of PCR products was done withthe aid of Molecular Analyst software (BioRad).

Example 5 Generation of Transgenic Mice

Murine TNFR25 constructs were cloned under the human CD2 promoter andlocal control region (gift from Dr. A. Singer, NIH) using therestriction endonuclease sites EcoRI and Sal I. Three mTNFR25 constructswere generated by PCR using a proofreading enzyme. The constructs werethe full length molecule of murine TNFR25 (FL TNFR25), the TNFR25 splicevariant lacking the 5th and 6th exon (Δ5,6 TNFR25), and the dominantnegative version of TNFR25 (DN TNFR25, as 1-234) terminating at the endof the transmembrane domain and lacking the entire intracellular domain.The sequence of the PCR products was confirmed by sequencing.Microinjections of DNA into the fertilized eggs were performed by thetransgenic facility at the University of Miami, School of Medicine.Potential founders were screened by PCR of DNA from tail biopsies. Theprimer pair was located upstream and downstream of the cloning sites,therefore the same primer pair was used for all the mTNFR25 transgenes.The upstream primer is 5′ CGC TCT TGC TCT CTG TGT ATG 3′ (SEQ ID NO: 14)and the downstream primer is 5′ CTG CCA GCC CTC TTC CAT C 3′ (SEQ ID NO:15). Transgenic mice were bred into the C57BL/6J background by seriallymating hemizygous transgenic animals with wild-type C57BL/6J (JacksonLaboratories, Bar Harbor, Me.). All mice were used at 6-12 weeks of ageand were maintained in pathogen-free facilities. The University of MiamiAnimal Care and Use Committee approved all animal use procedures.

Example 6 Nuclear Extract Preparation and Electrophoretic Mobility ShiftAssays for NFIKB Activation

One hundred and seven of EL4- Δ5,6 TNFR25 or EL4-FL TNFR25 cells weretreated with soluble or membrane bound TL1A or with the TNFR25 agonisticantibody 4C12 as indicated in the figure legends and then collected bycentrifugation at 800 g for 5 min. Nuclear extracts were isolated usinga minipreparation protocol and subjected to EMSA as described (Harhaj,E. W. et al. Virology 333, 145-58 (2005). Nuclear extracts (6 μg) wereincubated at room temperature for 20 min with a 32P-labeledhigh-affinity KB probe, followed by resolving the DNA-protein complexeson native 5% polyacrylamide gels.

Example 7 T Cell Proliferation Assay

Splenocytes were plated in triplicate at 1×105 cells/well in 96-wellflat-bottom plates. Cells were activated with immobilized anti-CD3 (2μg/ml) with or without soluble antiCD28 (1 μg/ml), or with ConA (5μg/ml) or with PMA (1 μg/ml) and ionomycin (400 ng/ml). For T cellproliferation, purified CD4 T cells at 1×105 cells/well or CD8 T cellsat 5×104 cells/well were stimulated with coated anti-CD3 (2 μg/ml) andsoluble anti-CD28 (1 μg/ml). Recombinant mIL-2 was added to the cultureat 1000 U/ml in indicated experiments. Cells were cultured for 72 hr andpulsed for the last 6 hours of incubation with 1 μCi/well of3H-thymidine (Perkin Elmer, Boston, Mass.), and thymidine incorporationwas quantitated using a scintillation counter.

Murine CD4 or CD8 or T cells were purified from splenocytes and/or lymphnodes by negative selection using SpinSep kit (StemCell Technology Inc.)according to the manufacturer's protocol. The purity was routinelyaround 90%-96% examined by F ACS analysis.

Example 8 Immunization of Mice with DNP-KLH, Antibody IsotypeDetermination, and Cytokine ELISA

Adult (6-10 wk old) transgenic and wt mice were immunizedintraperitoneally with 100 DNP-conjugated keyhole limpet hemocyanin(DNP-KLH) (CalBiochem). One week and three weeks after immunization,mice were bled and serum was separated for analysis of anti-DNP specificIgG1, IgE, and IgG2a antibodies by ELISA according to manufacturer'sprotocol (BD). Sera from individual animals were absorbed to 96-wellplates coated with 0.8 μg/ml DNP-albumin (DNP-BSA) (CalBiochem) and theisotype of bound antibody determined by ELISA.

To examine the cytokine production in the supernatants of cell cultures,sandwich ELISAs were performed per the manufacturer's instructions.Antibody pairs from BD were used for IL-2, IFN-γ, and IL-4 analysis.Reagents for IL-13 ELISA were purchased from R&D Systems (Minneapolis,Minn.) and reagents for IL-5 and IL-10 ELISA were purchased fromeBioscience.

Example 9 In Vitro Polarization CD4 T Cells into Th1 or Th2 Cells

CD4 T cells were purified by negative selection as mentioned above. CD4T cells were activated with immobilized anti-mCD3 (2 μg/ml) and solubleanti-mCD28 (1 μg/ml) alone, or in the presence of IL-12 (10 ng/ml) andanti-mIL-4 (20 μg/ml) for Th1 differentiation, or with IL-4 (10 ng/ml),anti-mIFN-γ (10 μg/ml), and anti-mIL-12 (10 μg/ml) for 7 days. The cellswere harvested, washed and replated at 1×105 cells per well andrestimulated with immobilized antiCD3. After 24 hours the supernatantswere harvested and evaluated for cytokine production by ELISA.

Example 10 Immunization Protocols for the Murine Model of AllergicAsthma

DN TNFR25-tg (encoded by SEQ ID NO: 16; FIG. 17D), Δ5,6 TNFR25-tg, andFL TNFR25-tg mice generated as described before and backcrossed at leastseven generations into the C57BL/6J background were compared withwild-type C57BL/6J mice purchased from National Cancer Institution(Frederick, Md.). Mice were sensitized by intraperitoneal injection of66 μg ovalbumin (crystallized chicken egg albumin, grade V; Sigma)absorbed to 6.6 mg aluminum potassium sulfate (alum; Sigma) in 200 μlPBS on day 0. On day 5, mice were boosted intraperitoneally with thesame dose of ovalbumin in alum. On day 12, mice were aerosol challengedwith 0.5% ovalbumin in PBS for one hour using an Ultrasonic Nebulizer(MABIS Healthcare Inc., Lake Forest, Ill.). Mice were assessed forallergic inflammation of the lungs three days after the single aerosolexposure. Mice were sacrificed by inhalation of CO2. After cannulationof the trachea the lung was lavaged 4 times with 1 ml of PBS. Cellsrecovered from the BAL fluid were counted and used for cytospinpreparations (50,000 cells or fewer/slide). >200 cells were counted foreach cytospin slide stained with Wright-Giemsa stain (Sigma) todetermine differential cell counts for macrophages, eosinophils,lymphocytes, and neutrophils.

Example 11 Lung Histology

Lungs were removed from mice after the bronchial lavage procedure andfixed in 10% neutral buffered formalin. Samples were submitted to theHistopathology Core of the Sylvester Cancer Center at the University ofMiami School of Medicine where specimens were embedded, sectioned, andstained with haematoxylin and eosin. Sections were also stained withperiodic acid-Schiff (PAS) to determine mucus production.

Example 12 ELISA for Serum Total IgE and Ovalbumin-Specific Ig

Mice were bled before sensitization (day 0), 3 days after aerosolchallenge (day 15) and in some experiments one day before aerosolchallenge (day 11). The total IgE level was quantitated by ELISAaccording to the manufacturer's protocol (BD). Ovalbumin specific IgEwas determined in Sandwich ELISA by first coating plate with 0.01% OVAin PBS, followed by loading diluted serum samples and then the secondarybiotin-labeled anti-IgE antibody (BD).

Example 13 In Vitro Restimulation of Bronchial Lymph Node Cells andCytokine Production

One day or three days after aerosol challenge, bronchial lymph nodeswere harvested and single cell suspensions were prepared. Cells wereseeded into round-bottom 96-well plates at 1×106 cells/well and culturedwith 100 μg/ml ovalbumin for 4 days. Then supernatants were collectedfor cytokine ELISA assays as described.

Example 14 Cytotoxicity Assay

Serially diluted soluble mTL1A supernatants harvested from P815-TL1Atransfected cells were added to 51Cr-labeled P815-TNFR25 transfectedtarget cells. To test for TL1A blocking activity different anti-TL1Amonoclonal antibodies were added into the culture and Cr releasedetermined after 4 hours in triplicate samples. Spontaneous release wascalculated from the wells that contained only 51Cr-labeled target cells.100% release (positive control) was calculated from the wells thatcontained 51 Cr-labeled target cells and 1% SDS. The percentage ofcytotoxicity activity was calculated as the following: (mean readout ofsample-mean readout of spontaneous release)/mean readout of positivecontrol. Similar data were also obtained with EL4-transfectants.

Example 15 Blocking of Lung Inflammation by Antagonistic Anti-mTL1AAntibody

Mice were sensitized intraperitoneally with ovalbumin in alum on day 0and day 5 followed by aerosol challenge with 0.5% ovalbumin in PBS forone hour on day 12. Mice were given L4G6 or an equivalent amount of thecontrol hamster IgG (Jackson Immuno Research) by intraperitonealinjection of 50 μg/mouse each day from day 11 to 14. Allergic lunginflammation was evaluated on day 15.

Example 16 Adoptive Transfer of NKT Cells

Ja18 k.o. mice (Cui, J. et al. Science 278, 1623-6 (1997)) were a giftfrom Michael Lotze (U. Pittsburgh) with kind permission from M.Taniguchi (Ciba University, Japan). NKT cells from w.t. and DN TNFR25-tgmice were isolated from pooled spleen cells from 10 mice by positiveselection using the EasySep mouse Pan NK Positive Selection Kit(StemCell Technologies, Vancouver, Canada) according to themanufacturer's instructions.

Example 17 Statistical Analyses

Statistical analyses using a two-tailed Student's t test were performedwith the GraphPad Prism Software (San Diego, Calif.); p<0.05 isconsidered significant. Data in the text are presented as the mean±SEM.

Example 18 Generation of DR3 and TL1A Antibodies

A DR3-Ig fusion protein was generated, purified and used to immunizehamsters. Hybridoma supernatants were obtained and screened by ELISAusing the DR3-Ig fusion protein as a screening agent. The nature of thehybridomas was verified by flow cytometry of DR3 transfected tumorcells, by Western blots, and by functional studies. All of theantibodies detected full-length and alternatively spliced DR3 ontransfected cells by F ACS, one of the antibodies detected DR3 inWestern blots, and the antibody (4C12) displayed agonistic activity,mediating DR3 signaling in the absence of TL1A.

TL1A monoclonal antibodies were obtained by immunizing hamsters with aTL1A-maltose-binding-protein fusion. The TL antibodies detectedtransfected TL1A by flow cytometry. The antibody (L4G6) displayedantagonistic activity, blocking TL1A binding to DR3.

Example 19 Signaling Through TNFR25 Enhances CD8 Cross-Priming

A novel heat shock protein gp96 based system that mediates strong,antigen specific CD8-CTL expansion in vivo was recently described inStrbo et al., Am J Reprod Immunol. 2002 October; 48(4):220-5. In thismodel system released gp96-Ig (engineered to be secreted) activatesdendritic cells and provides chaperoned peptides for cross presentationto and cross priming of CD8 cells (FIG. 12 ). The system is very usefulbecause it is independent of CD4 help. Secreted gp96 provides theactivation signal for DC through CD91 and TLR2/4 which is otherwiseprovided by CD40-L on CD4 cells. Accordingly, CD8 (OT-I) expansion inthis system works well in CD40-L deficient mice. This has been employedto study mucosal immunity and to determine the role of TNFR25 in CD8expansion.

EG7-gp96 is a cell line derived from the EL4 lymphoma by transfectionwith ovalbumin and gp96-Ig. The cells secrete gp96-Ig associated withovalbumin peptides. Ovalbumin-peptides chaperoned by gp96-Ig enhancecross-priming of CD8 cells (FIG. 10 ) by about 10,000 fold when comparedto ovalbumin alone. 100 ng ova-gp96-Ig expand OT-I in B6 mice from afrequency of 0.5% among CD8 cells post transfer to 20% in the spleenafter EG7-gp96-lg immunization.

In order to determine the effect of TNFR25 signals on CD8 expansion TCRtransgenic OT-I model as described above, were used together withEG7-gp96-Ig mediated stimulation. To determine the effect of TNFR25signals the mice received an agonistic anti TNFR25 antibody (4C12), aTNFR25 binding but not agonistic antibody (L4G6) or a control IgG 24hand 72 h after of EG7-gp96-Ig immunization. OT-I expansion wasmonitored in the peritoneal cavity on day 5 after immunization. 4C12caused an increased recruitment of cells into the peritoneal cavity byEG7-gp96-Ig resulting in a doubling of the cell number. In addition 4C12specifically caused an over 8-fold increase in the expansion of OT-I.The L4G6 anti TNFR25 antibody did not induce increased recruitment ofcells to the peritoneal cavity and inhibited OT-I expansion.

These data show that agonistic anti TNFR25 antibodies costimulate CD8cells and/or inhibit suppressive effects of Tregs via TNFR25.Costimulation of naive T cells by TNFR25 results in increasedproliferation and Th2 polarization upon secondary activation. Inaddition signaling of TNFR25 on T regulatory cells results in theirtemporary inhibition of suppression. The combined effect then isresponsible for the increased CD8 expansion and cell recruitment seen inFIGS. 11A and 11B.

Example 20 CD4+CD25+T Regulatory Cells Express High Levels on TNFR25

In order to determine the expression of TNFR25 CD4+CD25 Tregs werepurified from spleens by negative selection of CD4 cells followedmagnetic sorting with anti CD25. The cells were cultured with anti CD3,anti CD28 beads at a bead to cell ratio of 3:1 and 2000 u/ml human IL-2was added. Under these conditions the cells will begin to proliferateafter 3-4 days and keep expanding for about 3 weeks. The cultured cellswere analyzed by F ACS analysis for CD4 and CD25, by intracellularcytofluorimetry for FoxP3 expression and for surface analysis of TNFR25.FIG. 13 shows that Tregs obtained in this way are essentially pure andexpress both FoxP3 and TNFR25.

Example 21 TNFR25-Blockade Causes Lethality of Dextran Sodium SulfateColitis

The dextran sodium sulfate (DSS) model has widely been used as a colitismodel resembling in some aspects Crohn's disease. The initial insult isthe damaging effect of DSS on the permeability barrier normally providedby the gut epithelium. This effect of DSS allows access of the normalgut flora to sites in the mucosal immune system that set off aninflammatory immunological reaction resembling Crohn's colitis. Wildtype (w.t.) 136 mice during an 8 day course of exposure to drinkingwater containing 2% DSS develop diarrhea and lose weight. Uponrestoration of normal water, B6 mice recover and regain their normalweight. TNFR25 also influences the course of disease in Colitis. In thepresent experiment, w.t. and transgenic were exposed to mice to 2% DSSwater for seven days. As shown in FIG. 14 , DN-TNFR25-tg mice developeddisease similar to wild type mice, however when normal water wasrestored, DNTNFR25-tg mice did not recover as w.t. mice did. InsteadDN-TNFR25-tg mice continued to lose weight and died between day 12 and16. The Δ5,6-TNFR25-tg mice resembled w.t. mice although the death ofone mouse could also suggest a disturbed immune response. Twoconclusions were reached: In DNTNFR25-tg mice, the ensuing immuneresponse is much stronger than in w.t. mice leading to lethality and therestoration of normal health and homeostasis in w.t. mice is dependenton normally functioning T regulatory (Treg) cells. Treg function isdisturbed in DNTNFR25-tg mice. The latter is likely since it is knownthat Treg function is extraordinarily important to maintain homeostasisin the mucosal immune system by maintaining the correct balance betweentolerance to nutrients and normal gut flora and immune response to gutpathogens.

Example 22 Immunization with EG7-Gp96-Ig Induced the OT-I Cells toMigrate to Mucosal Sites Peyer's Patches, Lamina Propria Lymphocytes(LPL) and Intraepithelial Lymphocytes (IEL)

As shown in FIG. 16 (right column), EG7-cells are unable to cause clonalexpansion of OT-I or migration to mucosal sites. EG7-cells secretinggp96-Ig on the other hand cause clonal expansion of OT-I in spleen,lymph nodes and peritoneal cavity (not shown) and their migration tomucosal sites (FIG. 16 ). In Peyer's patches 8% of the cells are CD8+and 6.7% of the CDS-cells are OT-I; in IEL 61% of the cells are CD8+ and9% of the CD8 are OT-I. In LPL 29% of the CD8 cells are OT-I. The OT-Icells migrating to IEL after immunization are αEb7+ and α4β7+ but remainCD8αβ and TCRαβ, unlike resident CD8 IEL the majority of which are CD8aaand 50% TCRγδ.

In this disclosure there are described only the preferred embodiments ofthe invention and but a few examples of its versatility. It is to beunderstood that the invention is capable of use in various othercombinations and environments and is capable of changes or modificationswithin the scope of the inventive concept as expressed herein. Thus, forexample, those skilled in the art will recognize, or be able toascertain, using no more than routine experimentation, numerousequivalents to the specific substances and procedures described herein.Such equivalents are considered to be within the scope of thisinvention.

What is claimed is:
 1. A method for modulating antigen-specific T cellexpansion in a subject, the method comprising the steps of: (a)administering to the subject a cell expressing a foreign antigen; and(b) administering to the subject an amount of an agent whichspecifically binds TNFR25 effective to expand T cells specific for theforeign antigen in the subject.
 2. The method of claim 1, wherein theagent is an agonistic anti-TNFR25 antibody.
 3. The method of claim 1,wherein the agent is a soluble TL1A protein.
 4. The method of claim 1,wherein the cell is allogeneic to the subject.
 5. The method of claim 1,wherein the cell is a cancer cell.